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

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

Agency, qualia and life: connecting mind and body biologically

Many believe that a suitably programmed computer could act for its own goals and experience feelings. I challenge this view and argue that agency, mental causation and qualia are all founded in the unique, homeostatic nature of living matter. The theory was formulated for coherence with the concept of an agent, neuroscientific data and laws of physics. By this method, I infer that a successful action is homeostatic for its agent and can be caused by a feeling - which does not motivate as a force, but as a control signal. From brain research and the locality principle of physics, I surmise that qualia are a fundamental, biological form of energy generated in specialized neurons. Subjectivity is explained as thermodynamically necessary on the supposition that, by converting action potentials to feelings, the neural cells avert damage from the electrochemical pulses. In exchange for this entropic benefit, phenomenal energy is spent as and where it is produced - which precludes the objective observation of qualia

Adipocyte browning and higher mitochondrial function in peri-adrenal but not subcutaneous fat in pheochromocytoma

Context: Patients with pheochromocytoma (pheo) show presence of multilocular adipocytes that express uncoupling protein (UCP) 1 within periadrenal (pADR) and omental (OME) fat depots. It has been hypothesized that this is due to adrenergic stimulation by catecholamines produced by the pheo tumors. Objective: To characterize the prevalence and respiratory activity of brown-like adipocytes within pADR, OME and subcutaneous (SC) fat depots in human adult pheo patients. Design: This was an observational cohort study. Setting: University hospital. Patients: We studied 46 patients who underwent surgery for benign adrenal tumors (21pheos and 25 controls with adrenocortical adenomas). Main outcome measure: We characterized adipocyte browning in pADR, SC, and OME fat depots for histological and immunohistological features, mitochondrial respiration rate, and gene expression. We also determined circulating levels of catecholamines and other browning-related hormones. Results: 11 of 21 pheo pADR adipose samples, but only 1 of 25 pADR samples from control patients, exhibited multilocular adipocytes. The pADR browning phenotype was associated with higher plasma catecholamines and raised UCP1. Mitochondria from multilocular pADR fat of pheo patients exhibited increased rates of coupled and uncoupled respiration. Global gene expression analysis in pADR fat revealed enrichment in β-oxidation genes in pheo patients with multilocular adipocytes. No SC or OME fat depots exhibited aspects of browning. Conclusion: Browning of the pADR depot occurred in half of pheo patients and was associated with increased catecholamines and mitochondrial activity. No browning was detected in other fat depots, suggesting that other factors are required to promote browning in these depots

Individual variation in body temperature and energy expenditure in response to mild cold

Department of Human Biology, Maastricht University, 6200 MD Maastricht, The Netherlands. [email protected] We studied interindividual variation in body temperature and energy expenditure, the relation between these two, and the effect of mild decrease in environmental temperature (16 vs. 22 degrees C) on both body temperature and energy expenditure. Nine males stayed three times for 60 h (2000-0800) in a respiration chamber, once at 22 degrees C and twice at 16 degrees C, in random order. Twenty-four-hour energy expenditure, thermic effect of food, sleeping metabolic rate, activity-induced energy expenditure, and rectal and skin temperatures were measured. A rank correlation test with data of 6 test days showed significant interindividual variation in both rectal and skin temperatures and energy expenditures adjusted for body composition. Short-term exposure of the subjects to 16 degrees C caused a significant decrease in body temperature (both skin and core), an increase in temperature gradients, and an increase in energy expenditure. The change in body temperature gradients was negatively related to changes in energy expenditure. This shows that interindividual differences exist with respect to the relative contribution of metabolic and insulative adaptations to cold

Hydrogen-mediated photoelectrocatalysis with nickel-modified poly(heptazine imides)

Polymeric carbon nitrides (C3N4) are photochemically active organic semiconductors that can be produced in a wide range of structural types. Here, a poly-(heptazine imide) containing nickel single atoms (Ni-PHI) is employed for photochemical hydrogen production and is compared to the non-nickel-doped semiconductor. Film deposits are formed on a platinum disk electrode (to detect hydrogen) and a coating of the molecularly rigid polymer of intrinsic microporosity PIM-1 is applied to (i) mechanically stabilize the photo-catalyst film without impeding photocatalysis and (ii) assist in the interfacial hydrogen capture/oxygen suppression process. In the presence of hole quenchers such as methanol or ethanol, anodic photocurrents linked to hydrogen production/oxidation are observed. A comparison with an experiment on glassy carbon confirms the formation of interfacial hydrogen as a mediator. The effects of hole quencher concentration are evaluated. The system Pt/Ni-PHI/PIM-1 is employed in a single-compartment photo-fuel cell