Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest

Fabrication of a Horizontal and a Vertical Large Surface Area Nanogap Electrochemical Sensor

Nanogap sensors have a wide range of applications as they can provide accurate direct detection of biomolecules through impedimetric or amperometric signals. Signal response from nanogap sensors is dependent on both the electrode spacing and surface area. However, creating large surface area nanogap sensors presents several challenges during fabrication. We show two different approaches to achieve both horizontal and vertical coplanar nanogap geometries. In the first method we use electron-beam lithography (EBL) to pattern an 11 mm long serpentine nanogap (215 nm) between two electrodes. For the second method we use inductively-coupled plasma (ICP) reactive ion etching (RIE) to create a channel in a silicon substrate, optically pattern a buried 1.0 mm × 1.5 mm electrode before anodically bonding a second identical electrode, patterned on glass, directly above. The devices have a wide range of applicability in different sensing techniques with the large area nanogaps presenting advantages over other devices of the same family. As a case study we explore the detection of peptide nucleic acid (PNA)−DNA binding events using dielectric spectroscopy with the horizontal coplanar device

Maternal nutritional status in pastoral versus farming communities of West Pokot, Kenya: differences in iron and vitamin A status and body composition

BACKGROUND: Underweight and micronutrient deficiencies are sequelae of the prevailing harsh living and economic conditions of women in sub-Saharan Africa. There are few data describing maternal nutritional status in these resource-poor settings. Provision of more effective modes of intervention requires that public health and nutrition policy at both the national and the multisectoral levels be based on community-specific nutritional and behavioral practices. OBJECTIVE: This longitudinal study investigated maternal micronutrient status in two remote, semiarid, rural communities that are ethnically similar but have distinctly different pastoral and farming lifestyles. We looked at differences in iron stores, vitamin A levels, and body composition of women in the third trimester of pregnancy and again at 4 months postpartum. METHODS: Complete data were collected from 113 pastoral and 110 farming Pokot women. Anthropometric measurements were taken, and serum ferritin and retinol levels were measured. Infants were weighed within 7 days of birth. RESULTS: Women from the farming community had significantly (p < .05) lower hemoglobin concentrations than women from the pastoral community during the third trimester of pregnancy. Pastoral women had significantly higher serum ferritin concentrations than farming women during the third trimester of pregnancy (p <.05) and at 4 months postpartum. There were no significant differences between pastoral and farming women in the percentage of women with serum retinol levels < 0.70 micromol/L during the third trimester of pregnancy (27.9% [34/113] and 24.2% [31/110], respectively) and at 4 months postpartum (29.2% [33/113] and 30.9% [34/110]) In the farming community, mean infant birthweight was significantly lower (p <. 01) than in the pastoral community and a significantly higher (p < .05) proportion of newborns weighed less than 2.5 kg. At 4 months postpartum, the percentage of body fat was significantly lower in pastoral women than in farming women. CONCLUSIONS: Women from the farming community in West Pokot, Kenya, have lower iron stores during the third trimester of pregnancy than women in the pastoral community. In addition, the mean weight of their newborn infants is lower than that of infants in the pastoral community. These findings may be associated with differences in living conditions, which are usually harsher in farming than in pastoral communities. AD - Department of Epidemiology and Nutrition, School of Public Health, Moi University, Eldoret, Kenya. [email protected]

Sub-stoichiometric functionally graded titania fibres for water-splitting applications

The photo-electro-chemical (PEC) splitting of water requires semiconductor materials with a minimum energy gap of 1.23 eV along with conduction and valence bands overlapping the oxidation of H2O and reduction of H+ respectively. This work overcomes the limitations of stoichiometric titania by manufacturing fine scale fibres that exhibit a compositional gradient of oxygen vacancies across the fibre length. In such a fibre configuration the fibre end that is chemically reduced to a relatively small extent performs as the photoanode and the oxygen vacancies enhance the absorption of light. The fibre end that is reduced the most consists of Magnéli phases and exhibits metallic electrical conductivity that enhances the electron-hole separation. The structure and composition of the functionally graded fibres, which were manufactured through extrusion, pressureless sintering and carbo-thermal reduction, are studied using XRD and electron microscopy. Electrochemical impedance spectroscopy measurements were performed in a three-electrode electrochemical system and showed that the oxygen vacancies in the functionally graded fibres affect the flat band potential and have increased carrier density. The efficiency of the system was evaluated with PEC measurements that shows higher efficiency for the functionally graded fibres compared to homogeneous TiO2 or Magnéli phase fibres. The functionally graded and fine scale fibres have the potential to be used as an array of active fibres for water splitting applications.</p

Conductive Polymer-Coated 3D Printed Microneedles: Biocompatible Platforms for Minimally Invasive Biosensing Interfaces

\ua9 2023 The Authors. Small published by Wiley-VCH GmbH.Conductive polymeric microneedle (MN) arrays as biointerface materials show promise for the minimally invasive monitoring of analytes in biodevices and wearables. There is increasing interest in microneedles as electrodes for biosensing, but efforts have been limited to metallic substrates, which lack biological stability and are associated with high manufacturing costs and laborious fabrication methods, which create translational barriers. In this work, additive manufacturing, which provides the user with design flexibility and upscale manufacturing, is employed to fabricate acrylic-based microneedle devices. These microneedle devices are used as platforms to produce intrinsically-conductive, polymer-based surfaces based on polypyrrole (PPy) and poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS). These entirely polymer-based solid microneedle arrays act as dry conductive electrodes while omitting the requirement of a metallic seed layer. Two distinct coating methods of 3D-printed solid microneedles, in situ polymerization and drop casting, enable conductive functionality. The microneedle arrays penetrate ex vivo porcine skin grafts without compromising conductivity or microneedle morphology and demonstrate coating durability over multiple penetration cycles. The non-cytotoxic nature of the conductive microneedles is evaluated using human fibroblast cells. The proposed fabrication strategy offers a compelling approach to manufacturing polymer-based conductive microneedle surfaces that can be further exploited as platforms for biosensing

Genome engineering of isogenic human ES cells to model autism disorders.

Isogenic pluripotent stem cells are critical tools for studying human neurological diseases by allowing one to study the effects of a mutation in a fixed genetic background. Of particular interest are the spectrum of autism disorders, some of which are monogenic such as Timothy syndrome (TS); others are multigenic such as the microdeletion and microduplication syndromes of the 16p11.2 chromosomal locus. Here, we report engineered human embryonic stem cell (hESC) lines for modeling these two disorders using locus-specific endonucleases to increase the efficiency of homology-directed repair (HDR). We developed a system to: (1) computationally identify unique transcription activator-like effector nuclease (TALEN) binding sites in the genome using a new software program, TALENSeek, (2) assemble the TALEN genes by combining golden gate cloning with modified constructs from the FLASH protocol, and (3) test the TALEN pairs in an amplification-based HDR assay that is more sensitive than the typical non-homologous end joining assay. We applied these methods to identify, construct, and test TALENs that were used with HDR donors in hESCs to generate an isogenic TS cell line in a scarless manner and to model the 16p11.2 copy number disorder without modifying genomic loci with high sequence similarity

Impact of stirring regime on piezocatalytic dye degradation using BaTiO3 nanoparticles

There is increasing demand to use readily accessible waste energy to drive environmentally friendly processes. Piezocatalysis, the process of converting mechanical energy such as vibration into a chemical process, is a breakthrough next generation approach to meet this challenge. However, these systems currently focus on using ultrasound to drive the chemical reaction and are therefore expensive to operate. We show that by using simple mechanical stirring and BaTiO3 particles we can remove Rhodamine B dye molecules from solution. After evaluating a range of stirring parameters, we demonstrate that there is an interplay between stirring speed, volume of liquid, catalyst structure and rate of dye removal. Our maximum degradation rate was 12.05 mg. g-1 catalyst after 1 hour of mechanical stirring at favourable conditions. This development provides a new insight into a low energy physical technique that can be used in environmental remediation processes

Triphasic nature of polymers of intrinsic microporosity (PIM-1 and PIM-PY) induces storage and catalysis effects in hydrogen and oxygen reactivity at electrode surfaces

Hydrogen oxidation and oxygen reduction are two crucial energy conversion reactions, which are shown to be both strongly affected by the presence of intrinsically microporous polymer coatings on electrodes. Polymers of intrinsic microporosity (PIMs) are known to possess extremely high internal surface area and ability to bind gases under dry conditions. It is shown here that both, hydrogen‐ and oxygen gas binding into PIMs, also occurs under wet or “triphasic” conditions in aqueous electrolyte environments (when immersed in 0.01 M phosphate buffer at pH 7). For two known PIM materials (PIM‐1 and PIM‐PY), nanoparticles are formed by an anti‐solvent precipitation protocol and then cast as a film onto platinum or glassy carbon electrodes. Voltammetry experiments reveal evidence for hydrogen and oxygen binding. Both, PIM‐1 and PIM‐PY, locally store hydrogen or oxygen gas at the electrode surface and thereby significantly affect electrocatalytic reactivity. The onset of oxygen reduction on glassy carbon is shifted by 0.15 V in the positive direction

Giving electrons a ride: nanomechanical electron shuttles

Nanomechanical shuttles transferring small groups of electrons or even individual electrons from one electrode to another offer a novel approach to the problem of controlled charge transport. Here, we report the fabrication of shuttle-junctions consisting of a 20 nm diameter gold nanoparticle embedded within the gap between two gold electrodes. The nanoparticle is attached to the electrodes through a monolayer of flexible organic molecules which play the role of springs so that when a sufficient voltage bias is applied, then nanoparticle starts to oscillate transferring electrons from one electrode to the other. Current-voltage characteristics for the fabricated devices have been measured and compared with the results of our computer simulations.Comment: 11 pages, 4 figure