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1.  Size-dependent characteristics of electrostatically actuated fluid-conveying carbon nanotubes based on modified couple stress theory 
Summary
The paper presents the effects of fluid flow on the static and dynamic properties of carbon nanotubes that convey a viscous fluid. The mathematical model is based on the modified couple stress theory. The effects of various fluid parameters and boundary conditions on the pull-in voltages are investigated in detail. The applicability of the proposed system as nanovalves or nanosensors in nanoscale fluidic systems is elaborated. The results confirm that the nanoscale system studied in this paper can be properly applied for these purposes.
doi:10.3762/bjnano.4.88
PMCID: PMC3869342  PMID: 24367746
carbon nanotubes (CNT); electrostatic actuation; fluid flow; modified couple stress theory
2.  Block Copolymer Arrangement and Composition Effects on Protein Conformation Using AFM-Based Antigen-Antibody Adhesion 
The conformational changes of fibronectin deposited on various block copolymers where one block is composed of poly(methyl methacrylate) (PMMA) and the other block is either poly(acrylic acid) (PAA) or poly(2-hydroxyethyl methacrylate) (PHEMA) were investigated using a functionalized atomic force microscope (AFM) tip. The tip was modified with an antibody sensitive to the exposure of the arginine-glycine-aspartic acid (RGD) groups in fibronectin. By studying the adhesive interactions between the antibody and the proteins adsorbed on the block copolymer surface and phase imaging, it was found that the triblock copolymers PAA-b-PMMA-b-PAA and PMMA-b-PHEMA-b-PMMA, which both have large domain sizes, are conducive to the exposure of the fibronectin RGD groups on the surface. Based on these results, it is concluded that the surface chemistry as well as the nanomorphology dictated by the block copolymer arrangement could both tune protein conformation and orientation and optimize cell adhesion to the biomaterial surface.
doi:10.1002/jbm.a.34033
PMCID: PMC3677052  PMID: 22278846
block copolymers; atomic force microscopy; protein conformation; fibronectin; antibody
3.  Effect of spherical Au nanoparticles on nanofriction and wear reduction in dry and liquid environments 
Summary
Nano-object additives are used in tribological applications as well as in various applications in liquids requiring controlled manipulation and targeting. On the macroscale, nanoparticles in solids and liquids have been shown to reduce friction and wear. On the nanoscale, atomic force microscopy (AFM) studies have been performed in single- and multiple-nanoparticle contact, in dry environments, to characterize friction forces and wear. However, limited studies in submerged liquid environments have been performed and further studies are needed. In this paper, spherical Au nanoparticles were studied for their effect on friction and wear under dry conditions and submerged in water. In single-nanoparticle contact, individual nanoparticles, deposited on silicon, were manipulated with a sharp tip and the friction force was determined. Multiple-nanoparticle contact sliding experiments were performed on nanoparticle-coated silicon with a glass sphere. Wear tests were performed on the nanoscale with AFM as well as on the macroscale by using a ball-on-flat tribometer to relate friction and wear reduction on the nanoscale and macroscale. Results indicate that the addition of Au nanoparticles reduces friction and wear.
doi:10.3762/bjnano.3.85
PMCID: PMC3512125  PMID: 23213639
AFM; drug delivery; friction; gold nanoparticles; MEMS/NEMS; nanomanipulation
4.  Friction and durability of virgin and damaged skin with and without skin cream treatment using atomic force microscopy 
Summary
Skin can be damaged by the environment easily. Skin cream is an effective and rapid way to moisten the skin by changing the skin surface properties. Rat skin and pig skin are common animal models for studies and were used as skin samples in this study. The nano- and macroscale friction and durability of damaged skin were measured and compared with those of virgin (intact/undamaged) skin. The effect of skin cream on friction and durability of damaged and virgin skin samples is discussed. The effects of velocity, normal load, relative humidity and number of cycles were studied. The nanoscale studies were performed by using atomic force microscope (AFM), and macroscale studies were performed by using a pin-on-disk (POD) reciprocating tribometer. It was found that damaged skin has different mechanical properties, surface roughness, contact angle, friction and durability compared to that of virgin skin. But similar changes occur after skin cream treatment. Rat and pig skin show similar trends in friction and durability.
doi:10.3762/bjnano.3.83
PMCID: PMC3512123  PMID: 23213637
atomic force microscopy; damaged skin; pig skin; rat skin; skin cream
5.  Probing three-dimensional surface force fields with atomic resolution: Measurement strategies, limitations, and artifact reduction 
Summary
Noncontact atomic force microscopy (NC-AFM) is being increasingly used to measure the interaction force between an atomically sharp probe tip and surfaces of interest, as a function of the three spatial dimensions, with picometer and piconewton accuracy. Since the results of such measurements may be affected by piezo nonlinearities, thermal and electronic drift, tip asymmetries, and elastic deformation of the tip apex, these effects need to be considered during image interpretation.
In this paper, we analyze their impact on the acquired data, compare different methods to record atomic-resolution surface force fields, and determine the approaches that suffer the least from the associated artifacts. The related discussion underscores the idea that since force fields recorded by using NC-AFM always reflect the properties of both the sample and the probe tip, efforts to reduce unwanted effects of the tip on recorded data are indispensable for the extraction of detailed information about the atomic-scale properties of the surface.
doi:10.3762/bjnano.3.73
PMCID: PMC3458610  PMID: 23019560
atomic force microscopy; force spectroscopy; NC-AFM; three-dimensional atomic force microscopy; tip asymmetry; tip elasticity
6.  Fatty-Acid Binding Protein 4 Gene Polymorphisms and Plasma Levels in Children with Obstructive Sleep Apnea 
Sleep medicine  2011;12(7):666-671.
Introduction
Obstructive sleep apnea (OSA) is associated with increased risk for metabolic syndrome in both adults and children. In adults with OSA, serum levels of fatty acid binding protein 4 (FABP4) are elevated and associated with the degree of metabolic insulin resistance, independent of obesity. Therefore, we assessed plasma FABP4 levels and FABP4 allelic variants in obese and non-obese children with and without OSA.
Methods
A total of 309 consecutive children ages 5-8 years were recruited. Children were divided into those with OSA and without OSA (NOSA) based on the apnea-hypopnea index (AHI). Subjects were also subdivided into obese (OB) and non-obese (NOB) based on BMI z score). Morning fasting plasma FABP4 levels were assayed using ELISA, and 11 single-nucleotide polymorphisms (SNPs) within the FABP4 region were genotyped.
Results
Morning plasma FABP4 levels were increased in all children with OSA, even in NOB children. However, plasma FABP4 levels were strongly associated with BMI z score. Of the 11 SNPs tested, the frequency of rs1054135 (A/G) minor allele (A) was significantly increased in OSA. This SNP was also associated with increased plasma FABP4 levels in both OSA and obese subjects. The minor allele frequency of all other SNPs was similar in OSA and NOSA groups.
Conclusions
Childhood obesity and OSA are associated with higher plasma FABP4 levels and thus promote cardiometabolic risk. The presence of selective SNP (e.g., rs1054135) in the FABP4 gene may account for increased plasma FABP4 levels in the context of obesity and OSA in children.
doi:10.1016/j.sleep.2010.12.014
PMCID: PMC3144996  PMID: 21664182
8.  Integrative miRNA-mRNA Profiling of Adipose Tissue Unravels Transcriptional Circuits Induced by Sleep Fragmentation 
PLoS ONE  2012;7(5):e37669.
Obstructive sleep apnea (OSA) is a prevalent condition and strongly associated with metabolic disorders. Sleep fragmentation (SF) is a major consequence of OSA, but its contribution to OSA-related morbidities is not known. We hypothesized that SF causes specific perturbations in transcriptional networks of visceral fat cells, leading to systemic metabolic disturbances. We simultaneously profiled visceral adipose tissue mRNA and miRNA expression in mice exposed to 6 hours of SF during sleep, and developed a new computational framework based on gene set enrichment and network analyses to merge these data. This approach leverages known gene product interactions and biologic pathways to interrogate large-scale gene expression profiling data. We found that SF induced the activation of several distinct pathways, including those involved in insulin regulation and diabetes. Our integrative methodology identified putative controllers and regulators of the metabolic response during SF. We functionally validated our findings by demonstrating altered glucose and lipid homeostasis in sleep-fragmented mice. This is the first study to link sleep fragmentation with widespread disruptions in visceral adipose tissue transcriptome, and presents a generalizable approach to integrate mRNA-miRNA information for systematic mapping of regulatory networks.
doi:10.1371/journal.pone.0037669
PMCID: PMC3357342  PMID: 22629440
9.  Bioadhesion of various proteins on random, diblock and triblock copolymer surfaces and the effect of pH conditions 
The adhesive interactions of block copolymers composed of poly(methyl methacrylate) (PMMA)/poly(acrylic acid) (PAA) and poly(methyl methacrylate)/poly(2-hydroxyethyl methacrylate) (PHEMA) with the proteins fibronectin, bovine serum albumin and collagen were studied by atomic force microscopy. Adhesion experiments were performed both at physiological pH and at a slightly more acidic condition (pH 6.2) to model polymer–protein interactions under inflammatory or infectious conditions. The PMMA/PAA block copolymers were found to be more sensitive to the buffer environment than PMMA/PHEMA owing to electrostatic interactions between the ionized acrylate groups and the proteins. It was found that random, diblock and triblock copolymers exhibit distinct adhesion profiles although their chemical compositions are identical. This implies that biomaterial nanomorphology can be used to control protein–polymer interactions and potentially cell adhesion.
doi:10.1098/rsif.2010.0557
PMCID: PMC3061101  PMID: 21147831
block copolymers; atomic force microscopy; protein adhesion
10.  Associations of −308G/A Polymorphism of Tumor Necrosis Factor(TNF)–α Gene and Serum TNF-α Levels with Measures of Obesity, Intra-Abdominal and Subcutaneous Abdominal Fat, Subclinical Inflammation and Insulin Resistance in Asian Indians in North India 
Disease markers  2011;31(1):39-46.
Objectives: Obesity is associated with high levels proinflammatory cytokines like tumour necrosis factor alpha (TNF-α), which may play an important role in the genesis of insulin resistance. We evaluated the relationship of −308G/A polymorphism of TNF-α gene with obesity and insulin resistance in Asian Indians in north India.
Methods: This cross-sectional study included 151 apparently healthy individuals (79 males, 72 females) 18–50 yrs of age from New Delhi, India. Body composition by dual-energy x-ray absorptiometry (DEXA) and abdominal fat by magnetic resonance imaging (MRI) were measured. Biochemical measurements included OGTT, lipids, fasting insulin, hs-CRP and TNF-α levels. We analysed −308G/A polymorphism of TNF-α gene and studied its association with obesity and biochemical parameters.
Results: At comparable BMI, abdominal obesity was more prevalent in females (50%) as compared to males (20%). The wild genotype (GG) was present in 78.8%, GA in 17.9%, and AA in 3.3% subjects. Measures of body composition, abdominal fat distribution, lipids, insulin, hs-CRP and TNF-α levels were not influenced by the presence of −308G/A polymorphism. Serum TNF-α levels correlated significantly with fasting insulin in both genders.
Conclusion: TNF-α levels correlate with fasting insulin but not with indicators of body composition in Asian Indians. The −308G/A polymorphism of TNF-α gene is not associated with differences in the serum levels of TNF-α in Asian Indians.
doi:10.3233/DMA-2011-0802
PMCID: PMC3826921  PMID: 21846948
TNF-α gene polymorphism; obesity; abdominal fat; insulin resistance; Asian Indians
11.  Synthesis and Morphological Characterization of Block Copolymers for Improved Biomaterials 
Ultramicroscopy  2010;110(6):639-649.
Biocompatible polymers are known to act as scaffolds for the regeneration and growth of bone. Block copolymers are of interest as scaffold materials because a number of the blocks are biocompatible, and their nanostructure is easily tunable with synthetic techniques. In this paper, we report the synthesis of a novel class of biomaterials from block copolymers containing a hydrophobic block of methyl methacrylate and a hydrophilic block of either acrylic acid, dimethyl acrylamide, or 2-hydroxyethyl methacrylate. The block copolymers were synthesized using a combination of reversible addition-fragmentation chain transfer (RAFT) polymerization and click chemistry. Since the surface morphology is critical for successful cell growth, atomic force microscopy (AFM) studies were conducted for selected block copolymers. The topography, phase angle and friction maps were obtained in dry and physiological buffer environments to study the morphology. Results of AFM imaging identified the presence of polymer domains corresponding to the copolymer components. The distribution of nanoscale features in these block copolymers are comparable to those found on other surfaces that exhibit favorable cell adhesion and growth. In physiological buffer medium, the hydrophilic component of the block copolymer (acrylic acid or hydroxyethyl methacrylate) appear to be present in greater amounts on the surface as a consequence of water absorption and swelling.
doi:10.1016/j.ultramic.2010.02.025
PMCID: PMC2906250  PMID: 20207483
block copolymers; RAFT; click coupling; atomic force microscopy
12.  Association of PPARγ2 (Pro12Ala) and Neuropeptide Y (Leu7Pro) Gene Polymorphisms with Obstructive Sleep Apnea in Obese Asian Indians 
Disease markers  2011;30(1):31-38.
Background: Obstructive sleep apnea (OSA) is prevalent in 7.5% in urban Asian Indians. Peroxisome proliferator activated receptor gamma2 (PPARγ2) has been implicated in adipocyte differentiation. Neuropeptide Y (NPY) is also considered as a candidate gene for excess body fat accumulation. The association of PPARγ2 (Pro12Ala) and NPY (Leu7Pro) gene polymorphisms with OSA has not been studied in Asian Indians.
Objective: To study the distribution of PPARγ2 (Pro12Ala) and NPY (Leu7Pro) polymorphism in Asian Indians with and without OSA.
Methods and results: This study was carried out in 252 obese subjects [(body mass index (BMI > 25 kg/m2)]; 142 with OSA and 110 without OSA. Measurements included anthropometric and biochemical parameters (fasting blood glucose, lipid profile, various circumferences and skin-fold thicknesses). PPARγ2 (Pro12Ala) and NPY (Leu7Pro) gene polymorphisms were studied in all subjects. The frequency of the variant allele (Ala12) of PPARγ2 gene was significantly higher in subjects with OSA (14.4%) when compared with subjects without OSA (5.5%; χ2 = 9.7; p = 0.001). The distribution of the variant allele (Pro7) of NPY gene was comparable in subjects with OSA (3.5%) and without OSA (3.6%; χ 2 = 0.001, p = 0.94).
Conclusion: This study reveals a significantly higher frequency of PPARγ2 (Ala12) allele in obese Asian Indians with OSA when compared to obese Asian Indians without OSA.
doi:10.3233/DMA-2011-0762
PMCID: PMC3825082  PMID: 21508507
Obstructive sleep apnea; PPAR gamma; neuropeptide Y; gene polymorphism; Asian Indians; obesity
13.  Biomimetics inspired surfaces for drag reduction and oleophobicity/philicity 
Summary
The emerging field of biomimetics allows one to mimic biology or nature to develop nanomaterials, nanodevices, and processes which provide desirable properties. Hierarchical structures with dimensions of features ranging from the macroscale to the nanoscale are extremely common in nature and possess properties of interest. There are a large number of objects including bacteria, plants, land and aquatic animals, and seashells with properties of commercial interest. Certain plant leaves, such as lotus (Nelumbo nucifera) leaves, are known to be superhydrophobic and self-cleaning due to the hierarchical surface roughness and presence of a wax layer. In addition to a self-cleaning effect, these surfaces with a high contact angle and low contact angle hysteresis also exhibit low adhesion and drag reduction for fluid flow. An aquatic animal, such as a shark, is another model from nature for the reduction of drag in fluid flow. The artificial surfaces inspired from the shark skin and lotus leaf have been created, and in this article the influence of structure on drag reduction efficiency is reviewed. Biomimetic-inspired oleophobic surfaces can be used to prevent contamination of the underwater parts of ships by biological and organic contaminants, including oil. The article also reviews the wetting behavior of oil droplets on various superoleophobic surfaces created in the lab.
doi:10.3762/bjnano.2.9
PMCID: PMC3148050  PMID: 21977417
aquatic animals; biomimetics; drag; lotus plants; shark skin; superhydrophobicity; superoleophobicity
14.  Nanoscale adhesion, friction and wear studies of biomolecules on silane polymer-coated silica and alumina-based surfaces 
Proteins on biomicroelectromechanical systems (BioMEMS) confer specific molecular functionalities. In planar FET sensors (field-effect transistors, a class of devices whose protein-sensing capabilities we demonstrated in physiological buffers), interfacial proteins are analyte receptors, determining sensor molecular recognition specificity. Receptors are bound to the FET through a polymeric interface, and gross disruption of interfaces that removes a large percentage of receptors or inactivates large fractions of them diminishes sensor sensitivity. Sensitivity is also determined by the distance between the bound analyte and the semiconductor. Consequently, differential properties of surface polymers are design parameters for FET sensors. We compare thickness, surface roughness, adhesion, friction and wear properties of silane polymer layers bound to oxides (SiO2 and Al2O3, as on AlGaN HFETs). We compare those properties of the film–substrate pairs after an additional deposition of biotin and streptavidin. Adhesion between protein and device and interfacial friction properties affect FET reliability because these parameters affect wear resistance of interfaces to abrasive insult in vivo. Adhesion/friction determines the extent of stickage between the interface and tissue and interfacial resistance to mechanical damage. We document systematic, consistent differences in thickness and wear resistance of silane films that can be correlated with film chemistry and deposition procedures, providing guidance for rational interfacial design for planar AlGaN HFET sensors.
doi:10.1098/rsif.2008.0398
PMCID: PMC2839939  PMID: 18986962
adhesion; immunoFET; friction; silicon; AlGaN; heterojunction field-effect transistor sensors
15.  Prevalence of use of advance directives, health care proxy, legal guardian, and living will in 512 patients hospitalized in a cardiac care unit/intensive care unit in 2 community hospitals 
Introduction
The prevalence of use of any advance directives was 26% in 112 patients hospitalized in a cardiac care unit (CCU)/intensive care unit (ICU) in an academic medical center.
Material and methods
We investigated in 2 community hospitals the prevalence of use of advance directives (AD), health care proxy (HCP), legal guardian (LG), and living will (LW) in 512 patients hospitalized in a CCU/ ICU approached for AD and HCP.
Results
The use of AD was 22%, of HCP was 19%, of LG was 16%, and of LW was 5%.
Conclusions
The use of AD was 22%, of HCP was 19%, of LG was 16%, and of LW was 5% in patients hospitalized in a CCU/ICU. Educational programs on use of AD and of HCP need to be part of cardiovascular training programs and of cardiovascular continuing medical education.
doi:10.5114/aoms.2010.13892
PMCID: PMC3281338  PMID: 22371745
advance directives; health care proxy
16.  Fatty-acid binding protein 4 gene variants and childhood obesity: potential implications for insulin sensitivity and CRP levels 
Introduction
Obesity increases the risk for insulin resistance and metabolic syndrome in both adults and children. FABP4 is a member of the intracellular lipid-binding protein family that is predominantly expressed in adipose tissue, and plays an important role in maintaining glucose and lipid homeostasis. The purpose of this study was to measure FABP4 plasma levels, assess FABP4 allelic variants, and explore potential associations with fasting glucose and insulin levels in young school-age children with and without obesity.
Methods
A total of 309 consecutive children ages 5-7 years were recruited. Children were divided based on BMI z score into Obese (OB; BMI z score >1.65) and non-obese (NOB). Fasting plasma glucose, lipids, insulin, hsCRP, and FABP4 levels were measured. HOMA was used as correlate of insulin sensitivity. Four SNPs of the human FABP4 gene (rs1051231, rs2303519, rs16909233 and rs1054135), corresponding to several critical regions of the encoding FABP4 gene sequence were genotyped.
Results
Compared to NOB, circulating FABP4 levels were increased in OB, as were LDL, hsCRP and HOMA. FABP4 levels correlated with BMI, and also contributed to the variance of HOMA and hsCRP, but not serum lipids. The frequency of rs1054135 allelic variant was increased in OB, and was associated with increased FABP4 levels, while the presence of rs16909233 variant allele, although similar in OB and NOB, was associated with increased HOMA values.
Conclusions
Childhood obesity is associated with higher FABP4 levels that may promote cardiometabolic risk. The presence of selective SNPs in the FABP4 gene may account for increased risk for insulin resistance or systemic inflammation in the context of obesity.
doi:10.1186/1476-511X-9-18
PMCID: PMC2830195  PMID: 20156355
17.  The adhesion model considering capillarity for gecko attachment system 
Geckos make use of approximately a million microscale hairs (setae) that branch off into hundreds of nanoscale spatulae to cling to different smooth and rough surfaces and detach at will. This hierarchical surface construction gives the gecko the adaptability to create a large real area of contact with surfaces. It is known that van der Waals force is the primary mechanism used to adhere to surfaces, and capillary force is a secondary effect that can further increase adhesive force. To investigate the effects of capillarity on gecko adhesion, we considered the capillary force as well as the solid-to-solid interaction. The capillary force expressed in terms of elliptical integral is calculated by numerical method to cope with surfaces with a wide range of contact angles. The adhesion forces exerted by a single gecko spatula in contact with planes with different contact angles for various relative humidities are calculated, and the contributions of capillary force to total adhesion force are evaluated. The simulation results are compared with experimental data. Finally, using the three-level hierarchical model recently developed to simulate a gecko seta contacting with random rough surface, the effect of the relative humidity and the hydrophobicity of surface on the gecko adhesion is investigated.
doi:10.1098/rsif.2007.1078
PMCID: PMC2607397  PMID: 17594962
gecko; capillarity; adhesion; hierarchical structure
18.  Engineering functional protein interfaces for immunologically modified field effect transistor (ImmunoFET) by molecular genetic means 
The attachment and interactions of analyte receptor biomolecules at solid–liquid interfaces are critical to development of hybrid biological–synthetic sensor devices across all size regimes. We use protein engineering approaches to engineer the sensing interface of biochemically modified field effect transistor sensors (BioFET). To date, we have deposited analyte receptor proteins on FET sensing channels by direct adsorption, used self-assembled monolayers to tether receptor proteins to planar FET SiO2 sensing gates and demonstrated interface biochemical function and electrical function of the corresponding sensors. We have also used phage display to identify short peptides that recognize thermally grown SiO2. Our interest in these peptides is as affinity domains that can be inserted as translational fusions into receptor proteins (antibody fragments or other molecules) to drive oriented interaction with FET sensing surfaces. We have also identified single-chain fragment variables (scFvs, antibody fragments) that recognize an analyte of interest as potential sensor receptors. In addition, we have developed a protein engineering technology (scanning circular permutagenesis) that allows us to alter protein topography to manipulate the position of functional domains of the protein relative to the BioFET sensing surface.
doi:10.1098/rsif.2007.1107
PMCID: PMC2605506  PMID: 17580287
biosensors; interface engineering; surface modifications; protein engineering
19.  Towards optimization of patterned superhydrophobic surfaces 
Experimental and theoretical study of wetting properties of patterned Si surfaces with cylindrical flat-top pillars of various sizes and pitch distances is presented. The values of the contact angle (CA), contact angle hysteresis (CAH) and tilt angle (TA) are measured and compared with the theoretical values. Transition from the composite solid–liquid–air to the homogeneous solid–liquid interface is investigated. It is found that the wetting behaviour of a patterned hydrophobic surface depends upon a simple non-dimensional parameter, the spacing factor, equal to the pillar diameter divided by the pitch. The spacing factor controls the CA, CAH and TA in the composite interface regime, as well as destabilization and transition to the homogeneous interface. We show that the assumption that the CAH is a consequence of the adhesion hysteresis and surface roughness leads to the theoretical values of the CAH that are in a reasonably good agreement with the experimental values. By decreasing the spacing factor, the values of CA=170°, CAH=5° and TA=3° are achieved. However, with further decreasing of the spacing factor, the composite interface destabilizes.
doi:10.1098/rsif.2006.0211
PMCID: PMC2373387  PMID: 17251158
hydrophobic; lotus effect; contact angle; hysteresis; tilt angle
20.  Initial Reaction(s) in Biotransformation of CL-20 Is Catalyzed by Salicylate 1-Monooxygenase from Pseudomonas sp. Strain ATCC 29352 
CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane) (C6H6N12O12), a future-generation high-energy explosive, is biodegradable by Pseudomonas sp. strain FA1 and Agrobacterium sp. strain JS71; however, the nature of the enzyme(s) involved in the process was not understood. In the present study, salicylate 1-monooxygenase, a flavin adenine dinucleotide (FAD)-containing purified enzyme from Pseudomonas sp. strain ATCC 29352, biotransformed CL-20 at rates of 0.256 ± 0.011 and 0.043 ± 0.003 nmol min−1 mg of protein−1 under anaerobic and aerobic conditions, respectively. The disappearance of CL-20 was accompanied by the release of nitrite ions. Using liquid chromatography/mass spectrometry in the negative electrospray ionization mode, we detected a metabolite with a deprotonated mass ion [M − H]− at 345 Da, corresponding to an empirical formula of C6H6N10O8, produced as a result of two sequential N denitration steps on the CL- 20 molecule. We also detected two isomeric metabolites with [M − H]− at 381 Da corresponding to an empirical formula of C6H10N10O10. The latter was a hydrated product of the metabolite C6H6N10O8 with addition of two H2O molecules, as confirmed by tests using 18O-labeled water. The product stoichiometry showed that each reacted CL-20 molecule produced about 1.7 nitrite ions, 3.2 molecules of nitrous oxide, 1.5 molecules of formic acid, and 0.6 ammonium ion. Diphenyliodonium-mediated inhibition of salicylate 1-monooxygenase and a comparative study between native, deflavo, and reconstituted enzyme(s) showed that FAD site of the enzyme was involved in the biotransformation of CL-20 catalyzed by salicylate 1-monooxygenase. The data suggested that salicylate 1-monooxygenase catalyzed two oxygen-sensitive single-electron transfer steps necessary to release two nitrite ions from CL-20 and that this was followed by the secondary decomposition of this energetic chemical.
doi:10.1128/AEM.70.7.4040-4047.2004
PMCID: PMC444761  PMID: 15240281
21.  Biotransformation of 2,4,6,8,10,12-Hexanitro-2,4,6,8,10,12-Hexaazaisowurtzitane (CL-20) by Denitrifying Pseudomonas sp. Strain FA1 
The microbial and enzymatic degradation of a new energetic compound, 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20), is not well understood. Fundamental knowledge about the mechanism of microbial degradation of CL-20 is essential to allow the prediction of its fate in the environment. In the present study, a CL-20-degrading denitrifying strain capable of utilizing CL-20 as the sole nitrogen source, Pseudomonas sp. strain FA1, was isolated from a garden soil. Studies with intact cells showed that aerobic conditions were required for bacterial growth and that anaerobic conditions enhanced CL-20 biotransformation. An enzyme(s) involved in the initial biotransformation of CL-20 was shown to be membrane associated and NADH dependent, and its expression was up-regulated about 2.2-fold in CL-20-induced cells. The rates of CL-20 biotransformation by the resting cells and the membrane-enzyme preparation were 3.2 ± 0.1 nmol h−1 mg of cell biomass−1 and 11.5 ± 0.4 nmol h−1 mg of protein−1, respectively, under anaerobic conditions. In the membrane-enzyme-catalyzed reactions, 2.3 nitrite ions (NO2−), 1.5 molecules of nitrous oxide (N2O), and 1.7 molecules of formic acid (HCOOH) were produced per reacted CL-20 molecule. The membrane-enzyme preparation reduced nitrite to nitrous oxide under anaerobic conditions. A comparative study of native enzymes, deflavoenzymes, and a reconstituted enzyme(s) and their subsequent inhibition by diphenyliodonium revealed that biotransformation of CL-20 is catalyzed by a membrane-associated flavoenzyme. The latter catalyzed an oxygen-sensitive one-electron transfer reaction that caused initial N denitration of CL-20.
doi:10.1128/AEM.69.9.5216-5221.2003
PMCID: PMC194975  PMID: 12957905
22.  Biotransformation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) by a Rabbit Liver Cytochrome P450: Insight into the Mechanism of RDX Biodegradation by Rhodococcus sp. Strain DN22 
A unique metabolite with a molecular mass of 119 Da (C2H5N3O3) accumulated during biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Rhodococcus sp. strain DN22 (D. Fournier, A. Halasz, J. C. Spain, P. Fiurasek, and J. Hawari, Appl. Environ. Microbiol. 68:166-172, 2002). The structure of the molecule and the reactions that led to its synthesis were not known. In the present study, we produced and purified the unknown metabolite by biotransformation of RDX with Rhodococcus sp. strain DN22 and identified the molecule as 4-nitro-2,4-diazabutanal using nuclear magnetic resonance and elemental analyses. Furthermore, we tested the hypothesis that a cytochrome P450 enzyme was responsible for RDX biotransformation by strain DN22. A cytochrome P450 2B4 from rabbit liver catalyzed a very similar biotransformation of RDX to 4-nitro-2,4-diazabutanal. Both the cytochrome P450 2B4 and intact cells of Rhodococcus sp. strain DN22 catalyzed the release of two nitrite ions from each reacted RDX molecule. A comparative study of cytochrome P450 2B4 and Rhodococcus sp. strain DN22 revealed substantial similarities in the product distribution and inhibition by cytochrome P450 inhibitors. The experimental evidence led us to propose that cytochrome P450 2B4 can catalyze two single electron transfers to RDX, thereby causing double denitration, which leads to spontaneous hydrolytic ring cleavage and decomposition to produce 4-nitro-2,4-diazabutanal. Our results provide strong evidence that a cytochrome P450 enzyme is the key enzyme responsible for RDX biotransformation by Rhodococcus sp. strain DN22.
doi:10.1128/AEM.69.3.1347-1351.2003
PMCID: PMC150102  PMID: 12620815
23.  EYSENCK'S PERSONALITY QUESTIONNAIRE SCORES OF HEROIN ADDICTS IN INDIA 
Indian Journal of Psychiatry  1990;32(1):25-29.
SUMMARY
One hundred and ten male heroin addicts were administered ‘Eysenck Personality Questionnaire’ a self reporting measure. High scores on psychoticism, neuroticism and lie scale and low scores on extroversion in heroin addicts as compared to normal controls (n=50) were observed in this study which were comparable with those reported earlier. Further, it appears that high neuroticism scores are more consistent feature of heroin addicts than deviation on extraversion. Also an attempt to uncover epidemiological factors underlying heroin addiction has been made.
PMCID: PMC2989564  PMID: 21927422

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