The aim of this paper was to estimate the Ca content of the sarcoplasmic reticulum (s.r.) and to compare this with the amount of Ca which enters the cell via the calcium current in systole. The s.r. Ca content was measured electrophysiologically in voltage-clamped rat ventricular myocytes. Rapid application of caffeine produced a transient increase of [Ca2+]i which was accompanied by a transient inward Na-Ca exchange current. The integral of this current gives a measure of the Ca2+ pumped out of the cell by Na-Ca exchange. Ni2+ (5 mM) inhibited the current and decreased the rate of fall of [Ca2+]i to 32% of the control suggesting that Na-Ca exchange is responsible for 68% of Ca removal from the cytoplasm following the addition of caffeine. Correcting for the Na-Ca independent Ca removal suggests that the s.r. Ca content is equivalent to about 120 mumol per litre cell. Furthermore we estimate that, during systole, Ca entry into the cell via the sarcolemmal calcium current is equal to about 6% of the Ca content of the s.r.

Lung organogenesis requires precise timing and coordination to effect spatial organization and function of the parenchymal cells. To provide a systematic broad-based view of the mechanisms governing the dynamic alterations in parenchymal cells over crucial periods of development, we performed a single-cell RNA-sequencing time-series yielding 102,571 epithelial, endothelial and mesenchymal cells across nine time points from embryonic day 12 to postnatal day 14 in mice. Combining computational fate-likelihood prediction with RNA in situ hybridization and immunofluorescence, we explore lineage relationships during the saccular to alveolar stage transition. The utility of this publicly searchable atlas resource (www.sucrelab.org/lungcells) is exemplified by discoveries of the complexity of type 1 pneumocyte function and characterization of mesenchymal Wnt expression patterns during the saccular and alveolar stages – wherein major expansion of the gas-exchange surface occurs. We provide an integrated view of cellular dynamics in epithelial, endothelial and mesenchymal cell populations during lung organogenesis.

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These experiments show that the Na-Ca exchange accounts for 67% of the calcium removal not mediated by the sarcoplasmic reticulum. This is a smaller fraction than in rabbit cardiac cells and highlights the importance of the Ca-ATPase in the rat heart.

[Ca2+]i was measured using the fluorescent indicator indo 1 in voltage-clamped ferret and rat ventricular myocytes. The Ca2+ content of the sarcoplasmic reticulum (SR) was estimated from the integral of the Na(+)-Ca2+ exchange current activated by caffeine. Refilling of the SR after caffeine removal was enhanced by stimulation. As the systolic Ca2+ transient recovered, the integral of the L-type Ca2+ current decreased and that of the Na(+)-Ca2+ exchange tail current increased. For the early pulses, the gain of Ca2+ via the Ca2+ current is greater than the loss via the exchanger, and during steady state stimulation, the fluxes are equal. The difference in the integrals gives a measure of the net gain of cell Ca2+ with each pulse. When these are summed, the calculated gain of cell Ca2+ agrees well with the increase of SR Ca2+ produced by stimulation, as measured from the caffeine-evoked currents. There was a nonlinear relationship between SR Ca2+ content and the magnitude of the systolic Ca2+ transient such that at high SR Ca2+ content a given increase of content had a greater effect on the Ca2+ transient than did an increase at low SR content. In conclusion, the effects of systolic Ca2+ on the Ca2+ current and Na(+)-Ca2+ exchange current provide a means to regulate SR Ca2+ content and thence the systolic Ca2+ transient.

1. The experiments were performed on voltage-clamped cells in which intracellular calcium concentration ([Ca2+]i) was measured with the fluorescent indicator indo-1 (acetoxymethyl ester (AM) loading). When cells were stimulated with a short (100 ms) depolarizing pulse, following a rest, the magnitude of the first systolic calcium transient was greater than that in the steady state (rest potentiation) and decayed to its steady level over a few stimuli. If a longer pulse (800 ms) was used then the systolic calcium transient was either unaffected or increased in magnitude following a rest. During constant stimulation, if the length of the pulse is decreased, then the magnitude of the calcium transient decreased reversibly over several beats. 2. The calcium entry into the cell was measured from the integral of the inward calcium current and the efflux from the Na+-Ca2+ exchange current on repolarization. During the negative staircase the calcium current was approximately constant whilst the Na+-Ca2+ exchange current decayed in parallel with the systolic calcium transient. A net loss of calcium from the cell can be calculated from the extra Na+-Ca2+ exchange current following the initial pulses. 3. The application of caffeine produces a transient increase of both [Ca2+]i and an inward Nae-Ca2P exchange current. The integral of this current can be used to estimate the caffeine-releasable calcium content of the sarcoplasmic reticulum (SR), which decreases following stimulation with short compared to long pulses. This difference in SR calcium content is quantitatively similar to that estimated from the sarcolemmal currents.4. At a given membrane potential, the relationship between [Ca2+]i and current during the caffeine exposure can be used to estimate the Na+-Ca2+ exchange flux from the measured [Ca2+]i and thence the Na+-Ca2+ exchange flux during depolarization.5. For a long depolarizing pulse the extrusion of calcium from the cell on Na+-Ca2+ exchange is comparable to the entry on the calcium current. In contrast, for short pulses the extrusion of calcium on the Na+-Ca2+ exchange immediately after the pulse is greater than the entry during the pulse on the calcium current. 6. These results show that rest potentiation can be correlated with changes in the amount of calcium stored in the SR and this, in turn, can be accounted for by sarcolemmal fluxes.In cardiac muscle, the systolic rise of [Ca2+], results from the extracellular fluid must exactly balance the active both Ca entry into the cell from the extracellular fluid calcium efflux. Any imbalance between influx and efflux (primarily via the Ca current) and Ca release from the will lead to changes in the calcium content of the cell and sarcoplasmic reticulum (SR). The subsequent decrease of thence contractility until a new equilibrium is re-[Ca2+]j, which produces mechanical relaxation, depends on established. It is thought that this is responsible for the both the reuptake of calcium into the SR and active inotropic effects of, for example, changing the rate or tr...

The continued emergence and spread of infectious agents is of great concern, and systems biology approaches to infectious disease research can advance our understanding of host-pathogen relationships and facilitate the development of new therapies and vaccines. Molecular characterization of infectious samples outside of appropriate biosafety containment can take place only subsequent to pathogen inactivation. Herein, we describe a modified Folch extraction using chloroform/methanol that facilitates the molecular characterization of infectious samples by enabling simultaneous pathogen inactivation and extraction of proteins, metabolites, and lipids for subsequent mass spectrometry-based multi-omics measurements. This single-sample metabolite, protein and lipid extraction (MPLEx) method resulted in complete inactivation of clinically important bacterial and viral pathogens with exposed lipid membranes, including Yersinia pestis, Salmonella Typhimurium, and Campylobacter jejuni in pure culture, and Yersinia pestis, Campylobacter jejuni, and West Nile, MERS-CoV, Ebola, and influenza H7N9 viruses in infection studies. In addition, >99% inactivation, which increased with solvent exposure time, was also observed for pathogens without exposed lipid membranes including community-associated methicillin-resistant Staphylococcus aureus, Clostridium difficile spores and vegetative cells, and adenovirus type 5. The overall pipeline of inactivation and subsequent proteomic, metabolomic, and lipidomic analyses was evaluated using a human epithelial lung cell line infected with wild-type and mutant influenza H7N9 viruses, thereby demonstrating that MPLEx yields biomaterial of sufficient quality for subsequent multi-omics analyses. Based on these experimental results, we believe that MPLEx will facilitate systems biology studies of infectious samples by enabling simultaneous pathogen inactivation and multi-omics measurements from a single specimen with high success for pathogens with exposed lipid membranes.

We have studied the effects of two polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on spontaneous and electrically stimulated contractions in single, isolated ventricular myocytes from rat hearts. The frequency of spontaneous waves of calcium release and contraction (induced by elevation of the bathing calcium concentration) is reduced in the presence of EPA. At the same time the resting level of intracellular calcium falls, the resting cell length increases and the amplitude of shortening decreases. All these effects are reversed on removal of EPA. Imaging of the waves of calcium release shows that the amplitude and the rate of propagation of the wave is increased in EPA. Consistent with the increased amplitude, integration of the caffeine‐induced Na+‐Ca2+ exchange current (a measure of the sarcoplasmic reticulum (SR) calcium content) is increased by both EPA and DHA. EPA has a maintained negative inotropic effect on voltage clamped myocytes. This seems to be entirely due to inhibition of the L‐type calcium current. Smaller depolarising pulses in control conditions that elicit the same calcium current as in EPA also activate the same level of contraction. This is in spite of the increased SR calcium content in EPA. It is concluded that PUFAs have two effects on the SR; they reduce the availability of calcium for uptake and they inhibit the release mechanism. Both of these effects should lower the frequency of spontaneous waves of calcium release. As spontaneous release of calcium can initiate arrhythmias, some of the anti‐arrhythmic action of PUFAs must be exerted at the level of the SR.