Effects of nilotinib on leukaemia cells using vibrational microspectroscopy and cell cloning

Over the last few years, both synchrotron-based FTIR (S-FTIR) and Raman microspectroscopies have helped to better understand the effects of drugs on cancer cells. However, cancer is a mixture of cells with different sensitivity/resistance to drugs. Furthermore, the effects of drugs on cells produce both chemical and morphological changes, the latter could affect the spectra of cells incubated with drugs. Here, we successfully cloned sensitive and resistant leukaemia cells to nilotinib, a drug used in the management of leukaemia. This allowed both the study of a more uniform population and the study of sensitive and resistant cells prior to the addition of the drug with both S-FTIR and Raman microspectroscopies. The incubation with nilotinib produced changes in the S-FTIR and Raman spectra of both sensitive and resistant clones to nilotinib. Principal component analysis was able to distinguish between cells incubated in the absence or presence of the drug, even in the case of resistant clones. The latter would confirm that the spectral differences between the so-called resistant clonal cells prior to and after adding a drug might reside on those more or less sensitive cells that have been able to remain alive when they were collected to be studied with S-FTIR or Raman microspectroscopies. The data presented here indicate that the methodology of cell cloning can be applied to different types of malignant cells. This should facilitate the identification of spectral biomarkers of sensitivity/resistance to drugs. The next step would be a better assessment of sensitivity/resistance of leukaemia cells from patients which could guide clinicians to better tailor treatments to each individual patient

Physiological Oxygen Causes the Release of Volatile Organic Compounds from Human Pluripotent Stem Cells with Possible Roles in Maintaining Self-Renewal and Pluripotency

Human pluripotent stem cells (hPSCs) have widespread potential biomedical applications. There is a need for large-scale in vitro production of hPSCs, and optimal culture methods are vital in achieving this. Physiological oxygen (2% O2) improves key hPSCs attributes, including genomic integrity, viability, and clonogenicity, however, its impact on hPSC metabolism remains un-clear. Here, Selected Ion Flow Tube-Mass Spectrometry (SIFT-MS) was used to detect and quantify metabolic Volatile Organic Compounds (VOCs) in the headspace of hPSCs and their differentiated progeny. hPSCs were cultured in either 2% O2 or 21% O2. Media was collected from cell cultures and transferred into glass bottles for SIFT-MS measurement. The VOCs acetaldehyde and dimethyl sulfide (DMS)/ethanethiol were significantly increased in undifferentiated hPSCs compared to their differentiating counterparts, and these observations were more apparent in 2% O2. Pluripotent marker expression was consistent across both O2 concentrations tested. Transcript levels of ADH4, ADH5, and CYP2E1, encoding enzymes involved in converting ethanol to acetaldehyde, were upregulated in 2% O2, and chemical inhibition of ADH and CYP2E1 decreased acetaldehyde levels in hPSCs. Acetaldehyde and DMS/ethanethiol may be indicators of altered metabolism pathways in physiological oxygen culture conditions. The identification of non-destructive biomarkers for hPSC characterization has the potential to facilitate large-scale in vitro manufacture for future biomedical application.</jats:p

Evolution of Microscopic Localization in Graphene in a Magnetic Field from Scattering Resonances to Quantum Dots

Graphene is a unique two-dimensional material with rich new physics and great promise for applications in electronic devices. Physical phenomena such as the half-integer quantum Hall effect and high carrier mobility are critically dependent on interactions with impurities/substrates and localization of Dirac fermions in realistic devices. We microscopically study these interactions using scanning tunneling spectroscopy (STS) of exfoliated graphene on a SiO2 substrate in an applied magnetic field. The magnetic field strongly affects the electronic behavior of the graphene; the states condense into welldefined Landau levels with a dramatic change in the character of localization. In zero magnetic field, we detect weakly localized states created by the substrate induced disorder potential. In strong magnetic field, the two-dimensional electron gas breaks into a network of interacting quantum dots formed at the potential hills and valleys of the disorder potential. Our results demonstrate how graphene properties are perturbed by the disorder potential; a finding that is essential for both the physics and applications of graphene.Comment: to be published in Nature Physic

Adverse childhood experiences and mental health in young adults: a longitudinal survey

BACKGROUND: Adverse childhood experiences (ACEs) have been consistently linked to psychiatric difficulties in children and adults. However, the long-term effects of ACEs on mental health during the early adult years have been understudied. In addition, many studies are methodologically limited by use of non-representative samples, and few studies have investigated gender and racial differences. The current study relates self-reported lifetime exposure to a range of ACEs in a community sample of high school seniors to three mental health outcomes–depressive symptoms, drug abuse, and antisocial behavior–two years later during the transition to adulthood. METHODS: The study has a two-wave, prospective design. A systematic probability sample of high school seniors (N = 1093) was taken from communities of diverse socioeconomic status. They were interviewed in person in 1998 and over the telephone two years later. Gender and racial differences in ACE prevalence were tested with chi-square tests. Each mental health outcome was regressed on one ACE, controlling for gender, race/ethnicity, and SES to obtain partially standardized regression coefficients. RESULTS: Most ACEs were strongly associated with all three outcomes. The cumulative effect of ACEs was significant and of similar magnitude for all three outcomes. Except for sex abuse/assault, significant gender differences in the effects of single ACEs on depression and drug use were not observed. However, boys who experienced ACEs were more likely to engage in antisocial behavior early in young adulthood than girls who experienced similar ACEs. Where racial/ethnic differences existed, the adverse mental health impact of ACEs on Whites was consistently greater than on Blacks and Hispanics. CONCLUSION: Our sample of young adults from urban, socio-economically disadvantaged communities reported high rates of adverse childhood experiences. The public health impact of childhood adversity is evident in the very strong association between childhood adversity and depressive symptoms, antisocial behavior, and drug use during the early transition to adulthood. These findings, coupled with evidence that the impact of major childhood adversities persists well into adulthood, indicate the critical need for prevention and intervention strategies targeting early adverse experiences and their mental health consequences

Mapping Dirac quasiparticles near a single Coulomb impurity on graphene

The response of Dirac fermions to a Coulomb potential is predicted to differ significantly from how non-relativistic electrons behave in traditional atomic and impurity systems. Surprisingly, many key theoretical predictions for this ultra-relativistic regime have not been tested. Graphene, a two-dimensional material in which electrons behave like massless Dirac fermions, provides a unique opportunity to test such predictions. Graphene’s response to a Coulomb potential also offers insight into important material characteristics, including graphene’s intrinsic dielectric constant, which is the primary factor determining the strength of electron–electron interactions in graphene. Here we present a direct measurement of the nanoscale response of Dirac fermions to a single Coulomb potential placed on a gated graphene device. Scanning tunnelling microscopy was used to fabricate tunable charge impurities on graphene, and to image electronic screening around them for a Q = +1|e| charge state. Electron-like and hole-like Dirac fermions were observed to respond differently to a Coulomb potential. Comparing the observed electron–hole asymmetry to theoretical simulations has allowed us to test predictions for how Dirac fermions behave near a Coulomb potential, as well as extract graphene’s intrinsic dielectric constant: ε[subscript g] = 3.0±1.0. This small value of ε[subscript g] indicates that electron–electron interactions can contribute significantly to graphene properties.United States. Office of Naval Research. Multidisciplinary University Research Initiative (Award N00014-09-1-1066)United States. Dept. of Energy. Office of Science (Contract DE-AC02-05CH11231)National Science Foundation (U.S.) (Award DMR-0906539

Probing the action of a novel anti-leukaemic drug therapy at the single cell level using modern vibrational spectroscopy techniques

Acute myeloid leukaemia (AML) is a life threatening cancer for which there is an urgent clinical need for novel therapeutic approaches. A redeployed drug combination of bezafibrate and medroxyprogesterone acetate (BaP) has shown anti-leukaemic activity in vitro and in vivo. Elucidation of the BaP mechanism of action is required in order to understand how to maximise the clinical benefit. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, Synchrotron radiation FTIR (S-FTIR) and Raman microspectroscopy are powerful complementary techniques which were employed to probe the biochemical composition of two AML cell lines in the presence and absence of BaP. Analysis was performed on single living cells along with dehydrated and fixed cells to provide a large and detailed data set. A consideration of the main spectral differences in conjunction with multivariate statistical analysis reveals a significant change to the cellular lipid composition with drug treatment; furthermore, this response is not caused by cell apoptosis. No change to the DNA of either cell line was observed suggesting this combination therapy primarily targets lipid biosynthesis or effects bioactive lipids that activate specific signalling pathways

First-Principles Study of the Electronic and Magnetic Properties of Defects in Carbon Nanostructures

Understanding the magnetic properties of graphenic nanostructures is instrumental in future spintronics applications. These magnetic properties are known to depend crucially on the presence of defects. Here we review our recent theoretical studies using density functional calculations on two types of defects in carbon nanostructures: Substitutional doping with transition metals, and sp$^3$-type defects created by covalent functionalization with organic and inorganic molecules. We focus on such defects because they can be used to create and control magnetism in graphene-based materials. Our main results are summarized as follows: i)Substitutional metal impurities are fully understood using a model based on the hybridization between the $d$ states of the metal atom and the defect levels associated with an unreconstructed D$_{3h}$ carbon vacancy. We identify three different regimes, associated with the occupation of distinct hybridization levels, which determine the magnetic properties obtained with this type of doping; ii) A spin moment of 1.0 $\mu_B$ is always induced by chemical functionalization when a molecule chemisorbs on a graphene layer via a single C-C (or other weakly polar) covalent bond. The magnetic coupling between adsorbates shows a key dependence on the sublattice adsorption site. This effect is similar to that of H adsorption, however, with universal character; iii) The spin moment of substitutional metal impurities can be controlled using strain. In particular, we show that although Ni substitutionals are non-magnetic in flat and unstrained graphene, the magnetism of these defects can be activated by applying either uniaxial strain or curvature to the graphene layer. All these results provide key information about formation and control of defect-induced magnetism in graphene and related materials.Comment: 40 pages, 17 Figures, 62 References; Chapter 2 in Topological Modelling of Nanostructures and Extended Systems (2013) - Springer, edited by A. R. Ashrafi, F. Cataldo, A. Iranmanesh, and O. Or

Positional Cloning of a Type 2 Diabetes Quantitative Trait Locus; Tomosyn-2, a Negative Regulator of Insulin Secretion

We previously mapped a type 2 diabetes (T2D) locus on chromosome 16 (Chr 16) in an F2 intercross from the BTBR T (+) tf (BTBR) Lepob/ob and C57BL/6 (B6) Lepob/ob mouse strains. Introgression of BTBR Chr 16 into B6 mice resulted in a consomic mouse with reduced fasting plasma insulin and elevated glucose levels. We derived a panel of sub-congenic mice and narrowed the diabetes susceptibility locus to a 1.6 Mb region. Introgression of this 1.6 Mb fragment of the BTBR Chr 16 into lean B6 mice (B6.16BT36–38) replicated the phenotypes of the consomic mice. Pancreatic islets from the B6.16BT36–38 mice were defective in the second phase of the insulin secretion, suggesting that the 1.6 Mb region encodes a regulator of insulin secretion. Within this region, syntaxin-binding protein 5-like (Stxbp5l) or tomosyn-2 was the only gene with an expression difference and a non-synonymous coding single nucleotide polymorphism (SNP) between the B6 and BTBR alleles. Overexpression of the b-tomosyn-2 isoform in the pancreatic β-cell line, INS1 (832/13), resulted in an inhibition of insulin secretion in response to 3 mM 8-bromo cAMP at 7 mM glucose. In vitro binding experiments showed that tomosyn-2 binds recombinant syntaxin-1A and syntaxin-4, key proteins that are involved in insulin secretion via formation of the SNARE complex. The B6 form of tomosyn-2 is more susceptible to proteasomal degradation than the BTBR form, establishing a functional role for the coding SNP in tomosyn-2. We conclude that tomosyn-2 is the major gene responsible for the T2D Chr 16 quantitative trait locus (QTL) we mapped in our mouse cross. Our findings suggest that tomosyn-2 is a key negative regulator of insulin secretion

The Role of Friends’ Disruptive Behavior in the Development of Children’s Tobacco Experimentation: Results from a Preventive Intervention Study

Having friends who engage in disruptive behavior in childhood may be a risk factor for childhood tobacco experimentation. This study tested the role of friends’ disruptive behavior as a mediator of the effects of a classroom based intervention on children’s tobacco experimentation. 433 Children (52% males) were randomly assigned to the Good Behavior Game (GBG) intervention, a universal preventive intervention targeting disruptive behavior, and facilitating positive prosocial peer interactions. Friends’ disruptive behavior was assessed from age 7–10 years. Participants’ experimentation with tobacco was assessed annually from age 10–13. Reduced rates in tobacco experimentation and friends’ disruptive behavior were found among GBG children, as compared to controls. Support for friends’ disruptive behavior as a mediator in the link between intervention status and tobacco experimentation was found. These results remained after controlling for friends’ and parental smoking status, and child ADHD symptoms. The results support the role of friends’ disruptive behavior in preadolescents’ tobacco experimentation