Brain-Implantable Multifunctional Probe for Simultaneous Detection of Glutamate and GABA Neurotransmitters.

Glutamate (GLU) and gamma-aminobutyric acid (GABA) are neurotransmitters (NTs) with an essential role in signal transmission in the brain. Brain disorders, such as epilepsy, Alzheimer's and Parkinson's diseases, and traumatic brain injury can be linked to imbalances in the GLU-GABA homeostasis that occurs in sub-second to seconds time frames. Current measurement techniques can detect these two NT concentrations simultaneously only in vitro. The present work reports on the fabrication of a silicon multifunctional biosensor microarray probe for sub-second simultaneous GLU-GABA detection in real-time, with excellent analyte sensitivity and selectivity and in vivo capabilities. The novel Si probes feature four surface-functionalized platinum ultramicroelectrodes (UMEs) for simultaneous amperometric detection of GLU and GABA with a sentinel, and a built-in microfluidic channel for the introduction of neurochemicals in the proximity of the UMEs. The microchannel also allows functioning of an On-Demand In-situ Calibrator that runs in-situ biosensor calibration. The probe exhibited excellent robustness at insertion in agarose-gel brain-tissue-mimicking test, and remarkably high hydrogen peroxide sensitivity (a by-product of GLU-GABA enzyme biosensor) with values on the order of 5000 nA µM -1 cm -2 and maximum sensitivities of 204±15 nA µM -1 cm -2 and 37±7 nA µM -1 cm -2 for GLU and GABA, respectively. Furthermore, the limit of detection of the biosensors reached as low as 7 nM, 165 nM and 750 nM for H 2 O 2, GLU and GABA, respectively and a temporal resolution of hundreds of milliseconds during in vivo studies using freely moving rats.

Identification of Interactions between Sindbis Virus Capsid Protein and Cytoplasmic vRNA as Novel Virulence Determinants.

Alphaviruses are arthropod-borne viruses that represent a significant threat to public health at a global level. While the formation of alphaviral nucleocapsid cores, consisting of cargo nucleic acid and the viral capsid protein, is an essential molecular process of infection, the precise interactions between the two partners are ill-defined. A CLIP-seq approach was used to screen for candidate sites of interaction between the viral Capsid protein and genomic RNA of Sindbis virus (SINV), a model alphavirus. The data presented in this report indicates that the SINV capsid protein binds to specific viral RNA sequences in the cytoplasm of infected cells, but its interaction with genomic RNA in mature extracellular viral particles is largely non-specific in terms of nucleotide sequence. Mutational analyses of the cytoplasmic viral RNA-capsid interaction sites revealed a functional role for capsid binding early in infection. Interaction site mutants exhibited decreased viral growth kinetics; however, this defect was not a function of decreased particle production. Rather mutation of the cytoplasmic capsid-RNA interaction sites negatively affected the functional capacity of the incoming viral genomic RNAs leading to decreased infectivity. Furthermore, cytoplasmic capsid interaction site mutants are attenuated in a murine model of neurotropic alphavirus infection. Collectively, the findings of this study indicate that the identified cytoplasmic interactions of the viral capsid protein and genomic RNA, while not essential for particle formation, are necessary for genomic RNA function early during infection. This previously unappreciated role of capsid protein during the alphaviral replication cycle also constitutes a novel virulence determinant.

Barriers to early diagnosis of symptomatic breast cancer: a qualitative study of Black African, Black Caribbean and White British women living in the UK.

OBJECTIVES: Understanding barriers to early diagnosis of symptomatic breast cancer among Black African, Black Caribbean and White British women in the UK. DESIGN: In-depth qualitative interviews using grounded theory methods to identify themes. Findings validated through focus groups. PARTICIPANTS: 94 women aged 33-91 years; 20 Black African, 20 Black Caribbean and 20 White British women diagnosed with symptomatic breast cancer were interviewed. Fourteen Black African and 20 Black Caribbean women with (n=19) and without (n=15) breast cancer participated in six focus groups. SETTING: Eight cancer centres/hospital trusts in London (n=5), Somerset (n=1), West Midlands (n=1) and Greater Manchester (n=1) during 2012-2013. RESULTS: There are important differences and similarities in barriers to early diagnosis of breast cancer between Black African, Black Caribbean and White British women in the UK. Differences were influenced by country of birth, time spent in UK and age. First generation Black African women experienced most barriers and longest delays. Second generation Black Caribbean and White British women were similar and experienced fewest barriers. Absence of pain was a barrier for Black African and Black Caribbean women. Older White British women (≥70 years) and first generation Black African and Black Caribbean women shared conservative attitudes and taboos about breast awareness. All women viewed themselves at low risk of the disease, and voiced uncertainty over breast awareness and appraising non-lump symptoms. Focus group findings validated and expanded themes identified in interviews. CONCLUSIONS: Findings challenged reporting of Black women homogenously in breast cancer research. This can mask distinctions within and between ethnic groups. Current media and health promotion messages need reframing to promote early presentation with breast symptoms. Working with communities and developing culturally appropriate materials may lessen taboos and stigma, raise awareness, increase discussion of breast cancer and promote prompt help-seeking for breast symptoms among women with low cancer awareness.

In situ observation of the surface processes involved in dissolution from the cleavage surface of calcite in aqueous solution using combined scanning electrochemical-atomic force microscopy (SECM-AFM).

The surface processes involved in the initial stages of the proton-assisted dissolution of the calcite single crystal cleavage plane (1014) have been identified using a combined scanning electro-chemical-atomic force microscope (SECM-AFM). This instrument employs a platinum-coated AFM probe, which functions as an electrode as well as a high-resolution topographical sensor. Dissolution in this arrangement is effected by the local electrogeneration of protons, produced by oxidation of water at the probe electrode. By careful control of the applied potential, it is possible to vary the magnitude of the electrogenerated flux of protons from the probe towards the calcite surface. Crucially, by generating a small proton flux for short time periods (0.5 s) it is possible to observe and monitor the initial sites in the dissolution process. Topographical images were recorded in the same area of the surface both prior to and after inducing dissolution, as a function of the proton flux. At low proton fluxes, of the order of 1 nmol cm-2s-1 or less, the surface was observed to dissolve by the nucleation of monolayer deep pits, with densities of about 10(8) cm-2. These pits are likely to be formed at point vacancies or atomic (impurity, for example) defects in the crystal lattice. As the proton flux was increased (over two orders of magnitude), these same etch pits were found to open into wider macro-pits, with an outline morphology that reflected the crystallographic orientation of the surface. At the highest proton fluxes, dissolution from macroscopic step edges became significant.

Electrochemical imaging of diffusion through single nanoscale pores.

A combined scanning electrochemical-atomic force microscope (SECM-AFM) has been used to probe the diffusional transport of target electroactive solutes in isolated nanopores of a track-etched membrane. A polycarbonate membrane (100-nm-diam pore size) hydrated with an electrolyte solution, containing a redox-active probe molecule, such as IrCl6(3-) or Fe(phen)3(2+), functions as the model membrane system. The use of a mobile Pt-coated AFM probe enables individual solution-filled pores to be topographically identified. Analysis of the corresponding current images for the diffusion-limited oxidation of the redox mediator indicates that solution is largely confined to pores in the membrane. Moreover, the tip collector current response provides information on diffusion of the mediator through the pore. Force-distance tip approach and retract measurements allow the radius of contact between the electrochemical-AFM tip and solution confined within a pore at the point of pull-off to be estimated.