Brazil Nut Effect Drives Pattern Formation in Early Mammalian Embryos

ABSTRACTThe formation of three-dimensional ordered spatial patterns, which is essential for embryonic development, tissue regeneration, and cancer metastasis, is mainly guided by the chemical concentration gradient of morphogens. However, since no chemical concentration gradient has been observed in the early embryonic development (pre-implantation) of mammals, the pattern formation mechanism has been unsolved for a long time. During the second cell fate decision of mouse embryos, the inner cell mass (ICM) segregates into topographically regionalized epiblast (EPI) and primitive endoderm (PrE) layers. Here, we report that the segregation process of PrE/EPI precursors coincides with an emerged periodic expansion-contraction vibration of the blastocyst cavity, which induces phase transition in the ICM compartment to a higher fluidity state and generates directional tissue flows. By experiments and modeling, we demonstrate that the spatial segregation of PrE and EPI precursors is mediated by a “Brazil nut effect”-like viscous segregation mechanism in which PrE precursors with low affinity gradually migrate to the surface of ICM along with the tissue flow, while EPI precursors with high affinity remains inside ICM under cavity vibration. Artificially manipulation of the frequency and amplitude of cavity vibration could control the process of spatial separation as well as lineage specification of PrE/EPI. Furthermore, disruption of the cavity vibration in the initial stage after segregation could reverse the ICM cells back to a mixed state. Therefore, this study reveals a fundamental mechanism that guarantees the robustness of cell segregation and pattern formation without specific morphogens in early mammalian embryos. Our model also emphasizes a conserved function of cavity structure that widely exists in organisms as an energy reservoir and converter between different forms, such as chemical and mechanical energy.

Application of an online ion-chromatography-based instrument for gradient flux measurements of speciated nitrogen and sulfur

The dry component of total nitrogen and sulfur atmospheric deposition remains uncertain. The lack of measurements of sufficient chemical speciation and temporal extent make it difficult to develop accurate mass budgets and sufficient process level detail is not available to improve current air–surface exchange models. Over the past decade, significant advances have been made in the development of continuous air sampling measurement techniques, resulting with instruments of sufficient sensitivity and temporal resolution to directly quantify air–surface exchange of nitrogen and sulfur compounds. However, their applicability is generally restricted to only one or a few of the compounds within the deposition budget. Here, the performance of the Monitor for AeRosols and GAses in ambient air (MARGA 2S), a commercially available online ion-chromatography-based analyzer is characterized for the first time as applied for air–surface exchange measurements of HNO3, NH3, NH4+, NO3−, SO2 and SO42−. Analytical accuracy and precision are assessed under field conditions. Chemical concentrations gradient precision are determined at the same sampling site. Flux uncertainty measured by the aerodynamic gradient method is determined for a representative 3-week period in fall 2012 over a grass field. Analytical precision and chemical concentration gradient precision were found to compare favorably in comparison to previous studies. During the 3-week period, percentages of hourly chemical concentration gradients greater than the corresponding chemical concentration gradient detection limit were 86, 42, 82, 73, 74 and 69 % for NH3, NH4+, HNO3, NO3−, SO2 and SO42−, respectively. As expected, percentages were lowest for aerosol species, owing to their relatively low deposition velocities and correspondingly smaller gradients relative to gas phase species. Relative hourly median flux uncertainties were 31, 121, 42, 43, 67 and 56 % for NH3, NH4+, HNO3, NO3−, SO2 and SO42−, respectively. Flux uncertainty is dominated by uncertainty in the chemical concentrations gradients during the day but uncertainty in the chemical concentration gradients and transfer velocity are of the same order at night. Results show the instrument is sufficiently precise for flux gradient applications.

Application of an online ion chromatography-based instrument for gradient flux measurements of speciated nitrogen and sulfur

The dry component of total nitrogen and sulfur atmospheric deposition remains uncertain. The lack of measurements of sufficient chemical speciation and temporal extent make it difficult to develop accurate mass budgets and sufficient process level detail is not available to improve current air-surface exchange models. Over the past decade, significant advances have been made in the development of continuous air sampling measurement techniques, resulting with instruments of sufficient sensitivity and temporal resolution to directly quantify air-surface exchange of nitrogen and sulfur compounds. However, their applicability is generally restricted to only one or a few of the compounds within the de position budget. Here, the performance of the Monitor for AeRosols and GAses in ambient air (MARGA 2S), an commercially available on-line ion chromatography-based analyzer is characterized for the first time as applied for air-surface exchange measurements of HNO3, NH3, NH4+, NO3−, SO2 and SO42−. Analytical accuracy and precision are assessed under field conditions. Chemical concentrations gradient precision are determined at the same sampling site. Flux uncertainty measured by the aerodynamic gradient method is determined for a representative 3-week period in fall 2012 over a grass field. Analytical precision and chemical concentration gradient precision were found to compare favorably in comparison to previous studies. During the 3-week period, percentages of hourly chemical concentration gradients greater than the corresponding chemical concentration gradient detection limit were 86 %, 42 %, 82 %, 73 %, 74 %, and 69 % for NH3, NH4+, HNO3, NO3−, SO2, and SO42−, respectively. As expected, percentages were lowest for aerosol species, owing to their relatively low deposition velocities and correspondingly smaller gradients relative to gas phase species. Relative hourly median flux uncertainties were 31 %, 121 %, 42 %, 43 %, 67 %, and 56 % for NH3, NH4+, HNO3, NO3−, SO2, and SO42−, respectively. Flux uncertainty is dominated by uncertainty in the chemical concentrations gradients during the day but uncertainty in the chemical concentration gradients and transfer velocity are of the same order at night. Results show the instrument is sufficiently precise for flux gradient applications.

Chemical Sensing via Chemotaxis of Euglena gracilis Confined in an Isolated Micro-Aquarium

On-chip cytotoxicity sensing for liquid substances was investigated by using the microbial chemotaxis of Euglena gracilis. The Euglena cells were confined in a closed-type micro-aquarium in a PDMS microchip, and the micro-aquarium was isolated from two microchannels to flow test and reference liquid substances. Small molecules of liquids permeated into PDMS and diffused into the water in the micro-aquarium, and thus, the chemical concentration gradient of test liquids was built in the micro-aquarium. The negative chemotactic movements of Euglena cells were observed for ethanol down to 0.5% within 2-5 min after the injection of diluted ethanol into one of the separated microchannels (counter reference = pure water). On the other hand, when 0.5% H2O2was introduced as a test liquid (counter reference = pure water), the Euglena cells fell into continuous rotation instead of single step turning and/or straight forward swimming. As a result, total swimming activity in the micro-aquarium decreased even after H2O2flow was switched back to water. The observation shows that the types of cytotoxic effects can be identified through the cell movement analysis.

DEAE-Dextran Induced Increase of Membrane Permeability and Inhibition of Photosynthesis in Dunaliella parva

As a prerequisite for studies of the intracellular distribution of enzym es of the glycerol cycle in Dunaliella parva, the effect of the polycation DEAE-dextran on the permeability of membranes for various endogenous compounds and on photosynthesis was investigated. DEAE-dextran induces an increase of the permeability of membranes for low and high molecu­lar weight compounds: Under the influence of DEAE-dextran low molecular com pounds of the cells diffuse more rapidly along the chemical concentration gradient into the medium than macro-molecules. Furthermore enzymes of the cytoplasm do occur more rapidly in the outer medium than enzymes of the chloroplasts. Photosynthetic CO2 fixation is inhibited already at low DEAE-dextran concentrations. This in­ direct inhibition is due to an unspecific efflux of compounds of low molecular weight, such as inorganic cations, metabolites or nucleotides. At high DEAE-dextran concentrations photo­ synthetic electron transport is directly affected at the level of the thylakoids: Electron transport is inhibited between plastoquinone and photosystem I. The efflux characteristics of marker enzymes of DEAE-dextran treated D. parva cells show that under optimal experimental conditions this technique may be a suitable tool to get information about the intracellular distribution of enzym es of unknown localization in the alga.

CSF bicarbonate regulation in metabolic acidosis: role of HCO3- formation in CNS

In metabolic acidosis, cerebrospinal fluid bicarbonate content (CSF [HCO3-]) falls in parallel with reductions in CSF CO2 tension (PCO2), and the fall is minimal with isocapnia. Regulation of CSF HCO3- was therefore investigated during 6 h of isocapnic metabolic acidosis in dogs. One group received intraventricular injections of acetazolamide to inhibit the centrally located carbonic anhydrase, essential in central nervous system (CNS) HCO3- formation, while the control group received intraventricular saline. Plasma [HCO3-] was reduced by 10 meq/l with iv infusion of 0.2 N HCl. CSF [HCO3-] fell in the control group from 22.8 to 17.7 meq/l at 6 h, whereas in the acetazolamide group it fell from 22.9 to 13.0 meq/l. Brain ammonia content was 1,286 +/- 153 microgram/100 g in the controls and 666.2 +/- 103 microgram/100 g in the acetazolamide-treated group at 6 h. Therefore, some reduction in CSF [HCO3-] occurred during 6 h of isocapnic metabolic acidosis along the chemical concentration gradient between CSF and blood, but further falls, in CSF [HCO3-] were minimized by de novo, carbonic anhydrase-dependent HCO3- formation within the CNS. Some of H+ formed were buffered by the increase in brain ammonia. These central mechanisms contribute to local CNS H+ homeostasis in metabolic acidosis.