Loading fluorescent Ca²⁺ indicators into living cells

Small-molecule fluorescent Ca2+ reporters are the most widely used tools in the field of Ca2+ signaling. The excellent spatial and temporal resolution afforded by fluorescent reporters has driven the understanding of Ca2+ as a messenger in many different cell types. In many situations, the cellular loading and monitoring of fluorescent Ca2+ indicators is quite trivial. However, there are numerous pitfalls that require consideration to ensure that optimal data are recorded. Fluorescent Ca2+ indicators have carboxylic acid groups for binding of Ca2+. Because these “free-acid” forms of the indicators are hydrophilic they cannot readily cross cell membranes and need to be introduced into cells using techniques such as microinjection, pinocytosis, or diffusion from a patch pipette. However, the most convenient and widely used method for loading indicators into cells is as hydrophobic compounds in which the carboxylic acid groups are esterified (commonly as acetoxymethyl [AM] or acetate esters). The ester versions of the indicators permeate the plasma membrane. The Ca2+-sensitive, free-acid form of the indicator is liberated following hydrolysis of the ester groups by intracellular esterases

Stability of the Magnetic Monopole Condensate in three- and four-colour QCD

It is argued that the ground state of three- and four-colour QCD contains a monopole condensate, necessary for the dual Meissner effect to be the mechanism of confinement, and support its stability on the grounds that it gives the off-diagonal gluons an effective mass sufficient to remove the unstable ground state mode.Comment: jhep.cls, typos corrected, references added, some content delete

Review of geographical stocks of tropical dolphins (Stenella spp. and Delphinus delphis) in the eastern Pacific

Information on geographical variation is reviewed for Stenella attenuata, S. longirostris, S. coeruleoalba, and Delphinus delphis in the eastern tropical Pacific, and boundaries for potential management units are proposed. National Marine Fisheries Service and Inter-American Tropical Tuna Commission sighting records made from 1979 to 1983 which were outside boundaries used in a 1979 assessment were examined for validity. Tagging returns and morphological data were also analyzed. Several stock ranges are expanded or combined. Three management units are proposed for S. attenuata: the coastal, northern offshore, and southern offshore spoiled dolphins. Four management units are proposed for S. longirostris: the Costa Rican, eastern, northern whitebelly, and southern whitebelly spinner dolphins. Two provisional management units are proposed for S. coeruleoalba: the northern and southern striped dolphins. Five management units (two of which are provisional) are proposed for D. delphis: the Baja neritic, northern, central, southern, and Guerrero common dolphins. Division into management units was based on morphological stock differences and distributional breaks. (PDF file contains 34 pages.

Relative importance of auditing to the accounting profession: Is auditing a profit center?

https://egrove.olemiss.edu/dl_proceedings/1030/thumbnail.jp

Dissolved oxygen dynamics during a phytoplankton bloom in the Ross Sea polynya

The Ross Sea polynya is one of the most productive regions in the Southern Ocean. However, limited access and high spatio-temporal variability of physical and biological processes limit the use of conventional oceanographic methods to measure early season primary productivity. High-resolution observations from two Seagliders provide insights into the timing of a bloom in the southern Ross Sea polynya in December 2010. Changes in chlorophyll and oxygen concentrations are used to assess bloom dynamics. Using a ratio of dissolved oxygen to carbon, net primary production is estimated over the duration of the bloom showing a sensitive balance between net autotrophy and heterotrophy. The two gliders, observing spatially distinct regions during the same period, found net community production rates of -0.9±0.7 and 0.7±0.4 g C m-2 d-1. The difference highlights the spatial variability of biological processes and is probably caused by observing different stages of the bloom. The challenge of obtaining accurate primary productivity estimates highlights the need for increased observational efforts, particularly focusing on subsurface processes not resolved using surface or remote observations. Without an increased observational effort and the involvement of emerging technologies, it will not be possible to determine the seasonal trophic balance of the Ross Sea polynya and quantify the shelf's importance in carbon export

Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines

This protocol describes a highly reproducible antibody-based method that provides protein level and phosphorylation status information from nanogram quantities of protein cell lysate. Nanocapillary isoelectric focusing (cIEF) combines with UV-activated linking chemistry to detect changes in phosphorylation status. As an example application, we describe how to detect changes in response to tyrosine kinase inhibitors (TKIs) in the phosphorylation status of the adaptor protein ​CrkL, a major substrate of the oncogenic tyrosine kinase ​BCR-​ABL in chronic myeloid leukemia (CML), using highly enriched CML stem cells and mature cell populations in vitro. This protocol provides a 2.5 pg/nl limit of protein detection (<0.2% of a stem cell sample containing <104 cells). Additional assays are described for phosphorylated tyrosine 207 (pTyr207)-​CrkL and the protein tyrosine phosphatase ​PTPRC/​CD45; these assays were developed using this protocol and applied to CML patient samples. This method is of high throughput, and it can act as a screen for in vitro cancer stem cell response to drugs and novel agents