Permeabilized Cell Preparations Research Articles

Antimicrobials Targeted to the Replication-Specific DNA Polymerases of Gram-Positive Bacteria: Target Potential of dnaE

DNA polymerases pol IIIC and dnaE [i.e. pol IIIE] are essential for replicative DNA synthesis in low G:C Gram-positive eubacteria. Therefore, they have strong potential as targets for development of Gram-positive-selective antibacterial agents. This work has sought to extend to dnaE the recent discovery of antimicrobial agents based on pol IIIC-specific dGTP analogs. Compound 324C, a member of the same dGTP analog family, was found to be a potent and selective inhibitor of isolated dnaE in vitro. Surprisingly, 324C had no inhibitory effect in either intact Bacillus subtilis cells or in permeabilized cell preparations used to assess replicative DNA synthesis directly. It is proposed that the failure of 324C in the intact cell is a consequence of two major factors: (i) its template-dependent base pairing mechanism, and (ii) a specific subordinate role which dnaE apparently plays to pol IIIC. To generate an effective dnaE-selective inhibitor of replicative DNA synthesis in Gram-positive bacteria, it will likely be necessary to develop a molecule that attacks the enzyme's active site directly, without binding to template DNA.

Glucose Metabolism in Legionella pneumophila : Dependence on the Entner-Doudoroff Pathway and Connection with Intracellular Bacterial Growth

Glucose metabolism in Legionella pneumophila was studied by focusing on the Entner-Doudoroff (ED) pathway with a combined genetic and biochemical approach. The bacterium utilized exogenous glucose for synthesis of acid-insoluble cell components but manifested no discernible increase in the growth rate. Assays with permeabilized cell preparations revealed the activities of three enzymes involved in the pathway, i.e., glucokinase, phosphogluconate dehydratase, and 2-dehydro-3-deoxy-phosphogluconate aldolase, presumed to be encoded by the glk, edd, and eda genes, respectively. Gene-disrupted mutants for the three genes and the ywtG gene encoding a putative sugar transporter were devoid of the ability to metabolize exogenous glucose, indicating that the pathway is almost exclusively responsible for glucose metabolism and that the ywtG gene product is the glucose transporter. It was also established that these four genes formed part of an operon in which the gene order was edd-glk-eda-ywtG, as predicted by genomic information. Intriguingly, while the mutants exhibited no appreciable change in growth characteristics in vitro, they were defective in multiplication within eukaryotic cells, strongly indicating that the ED pathway must be functional for the intracellular growth of the bacterium to occur. Curiously, while the deficient glucose metabolism of the ywtG mutant was successfully complemented by the ywtG(+) gene supplied in trans via plasmid, its defect in intracellular growth was not. However, the latter defect was also manifested in wild-type cells when a plasmid carrying the mutant ywtG gene was introduced. This phenomenon, resembling so-called dominant negativity, awaits further investigation.

Preparation of Cells and Reagents for Flow Cytometry

Flow cytometry is widely used for analyzing the expression of cell surface and intracellular molecules (on a per cell basis), characterizing and defining different cell types in heterogeneous populations, assessing the purity of isolated subpopulations, and analyzing cell size and volume. This technique is predominantly used to measure fluorescence intensity produced by fluorescent-labeled antibodies or ligands that bind to specific cell-associated molecules. A procedure for direct and indirect staining of single-cell suspensions of lymphoid tissue or peripheral blood lymphocytes to detect cell surface membrane antigens is presented. In addition, support protocols present methods for fluorescence labeling of purified antibodies. A protocol for flow cytometric analysis of intracellular antigens in single-cell suspensions is also included. Alternate protocols describe intracellular staining of unfixed cells in the presence of a detergent and staining of nonviable cells to facilitate discrimination of dead cells in fixed or permeabilized cell preparations.

Mitochondrial activation directly triggers the exocytosis of insulin in permeabilized pancreatic beta-cells.

In the pancreatic beta-cell, insulin secretion is stimulated by glucose metabolism resulting in membrane potential-dependent elevation of cytosolic Ca2+ ([Ca2+]c). This cascade involves the mitochondrial membrane potential (delta psi[m]) hyperpolarization and elevation of mitochondrial Ca2+ ([Ca2+]m) which activates the Ca(2+)-sensitive NADH-generating dehydrogenases. Metabolism-secretion coupling requires unidentified signals, other than [Ca2+]c, possibly generated by the mitochondria through the rise in [Ca2+]m. To test this paradigm, we have established an alpha-toxin permeabilized cell preparation permitting the simultaneous monitoring of [Ca2+] with mitochondrially targeted aequorin and insulin secretion under conditions of saturating [ATP] (10 mM) and of clamped [Ca2+]c at substimulatory levels (500 nM). The tricarboxylic acid (TCA) cycle intermediate succinate hyperpolarized delta psi(m), raised [Ca2+]m up to 1.5 microM and stimulated insulin secretion 20-fold, without changing [Ca2+]c. Blockade of the uniporter-mediated Ca2+ influx into the mitochondria abolished the secretory response. Moreover, glycerophosphate, which raises [Ca2+]m by hyperpolarizing delta psi(m) without supplying carbons to the TCA cycle, failed to stimulate exocytosis. Activation of the TCA cycle with citrate evoked secretion only when combined with glycerophosphate. Thus, mitochondrially driven insulin secretion at permissive [Ca2+]c requires both a substrate for the TCA cycle and a rise in [Ca2+]m. Therefore, mitochondrial metabolism generates factors distinct from Ca2+ and ATP capable of inducing insulin exocytosis.

Regulation of cADP-ribose-induced Ca2+ Release by Mg2+ and Inorganic Phosphate

cADP-ribose (cADPr) has recently been shown to release Ca2+ from an intracellular store of permeabilized T lymphocyte cell lines (Guse, A. H., da Silva, C. P., Emmrich, F., Ashamu, G. A., Potter, B. V. L., and Mayr, G. W. (1995) J. Immunol. 155, 3353-3359). Using permeabilized Jurkat and HPB. ALL T lymphocytes, the effects of varying concentrations of inorganic phosphate and Mg2+ on cADPr-induced Ca2+ release were investigated. cADPr-induced Ca2+ release was dependent on the concentration of inorganic phosphate, showing very low Ca2+ release activity between 0.5 and 2 mM inorganic phosphate. At 4 to 5 mM inorganic phosphate, the cADPr-induced Ca2+ release was much more pronounced, reaching maximal values at 10 mM inorganic phosphate. The underlying mechanism for this stimulatory effect was an increased loading of the cADPr-sensitive Ca2+ store, which was demonstrated by enhanced resequestration of Ca2+ selectively into the cADPr-sensitive Ca2+ store. The free Mg2+ concentration also influenced cADPr-induced Ca2+ release in permeabilized cells: at 0 and 8.58 mM the release was nearly completely abolished, whereas at 1.06 mM maximal Ca2+ release by cADPr was observed. High performance liquid chromatographic analysis of exogenously added cADPr revealed that the catabolism of cADPr at varying Mg2+ and Pi concentrations had only minor relevance for the modulatory effects observed. To correlate the effects of inorganic phosphate and Mg2+ on cADPr-induced Ca2+ release observed in the permeabilized cell preparations, measurements of these ions in intact Jurkat T lymphocytes were carried out. Intact Jurkat T cells stimulated via the T cell receptor middle dotCD3 complex did not respond with significant elevation of the free intracellular Mg2+ concentration. In contrast, stimulation via the T cell receptor middle dotCD3 complex resulted in an increase in the intracellular inorganic phosphate concentration. These data indicate a role for the intracellular inorganic phosphate concentration in the regulation of cADPr-mediated Ca2+ release in T lymphocytes.

Pharmacologic Characterization of the Inositol Trisphosphate Receptor in Rat Pancreas

Inositol 1,4,5-trisphosphate (InsP3) is an intracellular second messenger, produced upon stimulation of the phosphoinositide system, capable of mobilizing calcium from intracellular stores. The properties of InsP3 receptor sites in the rat pancreas were evaluated by binding studies with InsP3 labeled with 3H. Specific binding was very sensitive to pH variations between 7 and 9. Kinetic studies showed that specific binding of InsP3 at 0 degrees C was half-maximal in about 10 min and reached a plateau within 60 min with a K+1 = 3.37 x 10(9) mol-1min-1. Binding was reversible as addition of 10(-6) M unlabeled InsP3 was followed by dissociation of the bound ligand with a K-1 = 0.016 min-1. Scatchard analysis of the binding data was consistent with a single set of high-affinity sites with KD of 9.9 +/- 2.47 nM and a maximal binding capacity of 210 +/- 55 fmol/mg of protein (n = 7). The specificity of [3H]InsP3 binding to these sites was illustrated by the much weaker affinity for structural analogs such as inositol 1,3,4,5-tetrakisphosphate, phytic acid, fructose 1,6-bisphosphate, and heparin. To assess the functional relevance of the InsP3 binding sites, the Ca(2+)-releasing activity of InsP3 was measured in permeabilized acinar cell preparations. In the presence of oligomycin (10 micrograms/ml), Ca2+ movements were monitored with the fluorescent indicator fura-2 (free acid). Under these conditions, 4 mM ATP caused rapid uptake of Ca2+ by the vesicular component of the acinar cells. Addition of InsP3 (0.1-15 microM) caused a dose-dependent release of Ca2+ with half-maximal effect at 3.2 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Conditional activation of cAMP signal transduction by protein kinase C. The effect of phorbol esters on adenylyl cyclase in permeabilized and intact cells.

To understand the convergence of cAMP and protein kinase C signal transduction, adenylyl cyclase isozyme identification and biochemical studies were performed on the HT4 neural cell line. In HT4 cells, basal cAMP production by adenylyl cyclase types I and VI were unaffected by phorbol esters, nor did phorbol esters have any effect on forskolin-induced cAMP production. However, phorbol esters synergistically increased cAMP production when adrenaline receptors were simultaneously activated, indicating a conditional activation of cAMP production by phorbol esters. A permeabilized cell preparation was used to analyze the mechanism by which phorbol esters were affecting cAMP production. This preparation enables G-proteins to be activated directly by GTP gamma S or bacterial toxins. In the permeabilized cell preparation, phorbol esters enhanced cAMP produced by GTP gamma S-activated G-protein. A stimulatory G-protein pathway may be involved since phorbol esters synergistically increased cAMP production by cholera toxin, yet had no effect on that produced by pertussis toxin. In this cell culture model, phorbol esters stimulate cAMP production only when some component of the cAMP cascade is simultaneously activated. Moreover, the pattern of modulation suggests that protein kinase C acts on an activated component of the second messenger system, such as the G-protein or the coupling of the G-protein with adenylyl cyclase, and not on the resting state of the protein components.

Chemotactic peptide stimulation of arachidonic acid release in HL60 cells, an interaction between G protein and phospholipase C mediated signal transduction

The mechanism of phospholipase A 2 activation by chemotactic peptide was investigated in human promyelocytic HL60 cells. N-Formyl-methionyl-leucyl-phenylalanine (fMetLeuPhe) and the non-Lydrolyzable GTP analogue guanosine 5′-[γ-thio]triphosphate (GTP[S]) induced arachidonic acid release in permeabilized and metabolically inhibited HL60 cells, a preparation in which calcium was buffered and inositol phospholipid hydrolysis was inhibited. Inositol phosphate generation and arachidonic acid were shown to be temporally dissociated. These results suggest that receptor-dependent phospholipase C activity is not required for fMetLeuPhe to induce arachidonic acid release. However, fMetLeuPhe effects were highly calcium-dependent and inhibition of phospholipase C reduced fMetLeuPhe stimulation of arachidonic acid release even in the permeabilized cell preparation. We conclude that although phospholipase A 2 activation is linked to the fMetLeuPhe receptor independent of phospholipase C, actions of phospholipase C to mobilize calcium and release diacylglycerol may be important to phospholipase A 2 activation in the intact cell.

Relationship between arachidonate release and exocytosis in permeabilized human neutrophils stimulated with formylmethionyl-leucyl-phenylalanine (fMetLeuPhe), guanosine 5′-[γ-thio]triphosphate (GTP[S]) and Ca2+

The role of two G-proteins, Gp and Ge, in the stimulus-secretion pathway has been proposed on the basis of studies where GTP analogues have been introduced into permeabilized cell preparations. In this study, evidence is provided that two G-proteins are also involved when a receptor-directed agonist is used. Intact human neutrophils were made refractory to formylmethionyl-leucyl-phenylalanine (fMetLeuPhe) stimulation by metabolic inhibition and then permeabilized with streptolysin O to compare the intracellular requirements for exocytosis from specific and azurophilic granules and arachidonate release. In the presence of 1 microM-Ca2+ and 1 mM-MgATP, fMetLeuPhe or guanosine 5'-[gamma-thio]triphosphate (GTP[S]) induce secretion from both granule types as well as arachidonate release. Secretion and arachidonate release owing to fMetLeuPhe can occur in the absence of ATP, conditions under which G-protein-mediated activation of phospholipase C is suppressed. GTP[S]-induced secretion can also occur in the absence of MgATP, but GTP[S]-induced arachidonate release cannot. It is concluded that fMetLeuPhe, like GTP[S], stimulates secretion by interacting with another G-protein-mediated reaction apart from Gp. Evidence is provided that a possible target for the second G-protein-mediated reaction involved in fMetLeuPhe-induced secretion (but not GTP[S]-induced secretion) is phospholipase A2.

Permeable Cell Models in Stimulus-Secretion Coupling

A number of mechanical and chemical methods have been developed to achieve selective permeabilization of the plasma membrane. These methods have been applied to many cell types and have proven to be highly useful for studying stimulus-secretion coupling mechanisms. Thus far, this approach has contributed significantly to our understanding of phosphoinositide metabolism and the regulation of intracellular calcium. The permeabilization techniques also have contributed important information regarding cAMP-dependent pathways. In addition to studies of stimulus-secretion coupling, permeabilized cell preparations can be employed for investigations of enzyme activity in situ and the properties of intracellular organelles in general. Since cell permeabilization, particularly with chemical agents, is surprisingly easy, these techniques should find wide application for future studies.

Allosteric regulation of the state of adenylylation of glutamine synthetase in permeabilized cell preparations of Rhodopseudomonas sphaeroides.

Following a freeze-thaw cycle, and the treatment of Rhodopseudomonas sphaeroides with the nonionic detergent Lubrol PX, the permeabilized cell suspensions can be assayed directly both for the intracellular levels of glutamine synthetase and the state of adenylylation (i.e. the average number n of adenylylated subunits/dodecameric molecules). It seems that all components of the bicycle system are retained if cells grown with low concentrations of ammonia as the sole nitrogen source are used. The value of n was dependent upon the concentration of substrates (ATP, Pi) and allosteric effectors (ATP, glutamine and alpha-ketoglutarate) of adenylytransferase. The value of n affected by UTP, the specific substrate of the uridylyltransferase shows first the evidence that the bicycle cascade control system studied in Escherichia coli may exist in this phototrophic bacterium.

Guanine nucleotide-dependent inositol trisphosphate formation in chick heart cells.

Stimulation of muscarinic receptors in dissociated embryonic chick heart cells promotes the hydrolysis of the phosphoinositides resulting in accumulation of the breakdown products inositol trisphosphate, bisphosphate, and monophosphate (InsP3, Insp2, and InsP, respectively). [3H]InsP3 and [3H]InsP2 are significantly elevated within 10 seconds of carbachol addition, while there is a lag in the accumulation of [3H]InsP. The time courses of the formation of the inositol phosphates suggest that carbachol activates a polyphosphoinositide-specific phospholipase C resulting in the formation of InsP3, which is subsequently metabolized to InsP2 and InsP. High-performance liquid chromotography analysis demonstrates the formation of both naturally occurring InsP3 isomers (Ins-1,4,5-P3 and Ins-1,3,4,-P3) and of inositol tetrakisphosphate (InsP4) as well. To investigate whether a guanine nucleotide-binding protein couples receptor stimulation to phosphoinositide (PI) hydrolysis in the heart, we developed a saponin-permeabilized cell preparation that would allow external manipulation of the intracellular guanosine triphosphate (GTP) concentration. In the permeabilized cell preparation, guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) stimulates the accumulation of [3H]InsP, [3H]InsP2, [3H]InsP3, and [3H]InsP4. The effect of GTP gamma S is half-maximal at 1 microM and maximal above 100 microM. In contrast, GTP gamma S is ineffective in promoting PI hydrolysis in the nonpermeabilized cell except at high concentrations. Other guanine nucleotides also lead to the accumulation of [3H]InsP in the permeabilized cell, while 5'-adenylylimidodiphosphate does not. Carbachol also stimulates PI hydrolysis in the permeabilized cell preparation although it is less effective than in the intact cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Specificity of inositol phosphate-stimulated Ca2+ mobilization from Swiss-mouse 3T3 cells.

Pure samples of inositol 1,3,4-trisphosphate, inositol 1,3,4,5-tetrakisphosphate and inositol 1,2-cyclic 4,5-trisphosphate were prepared and tested for their ability to mobilize calcium from intracellular stores in a permeabilized Swiss mouse 3T3 cell preparation. In this system inositol 1,4,5-trisphosphate mobilizes Ca2+ with a half-maximal dose of 0.3 microM. Inositol 1,2-cyclic 4,5-trisphosphate mobilized Ca2+ to the same extent with a half-maximal dose of 0.3 microM, whereas inositol 1,3,4-trisphosphate required a half-maximal dose of approx. 9 microM to give the same effect. Inositol 1,3,4,5-tetrakisphosphate was ineffective up to 20 microM and at that concentration did not antagonize the mobilization induced by inositol 1,4,5-trisphosphate. The relevance of these findings to the function of the inositol tris/tetrakis-phosphate pathway is discussed.

ATP‐induced cell contraction in dermal fibroblasts: Effects of cAMP and myosin light‐chain kinase

When 1 mM ATP is added to human dermal fibroblasts (DF) in monolayer culture permeabilized by glycerol, they undergo a rapid reduction in length and their intracellular actin filaments aggregate. This process is referred to as cell contraction. Treating glycerol-permeabilized DF with alkaline phosphatase before adding 1 mM ATP should cause dephosphorylation. Dephosphorylated preparations do not undergo cell contraction initiated by ATP. When myosin light-chain kinase (MLCK) isolated from turkey gizzard is added with cofactors to cells dephosphorylated by alkaline phosphatase treatment, contraction is restored. DF incubated for 24 h with db cAMP or cholera toxin show elevated intracellular concentrations of cAMP and little cell contraction. Contraction is reestablished when MLCK with cofactors is incubated with these preparations before ATP is added. Fibroblasts from Epidermolysis Bullosa dystrophica recessive patients produce excess cAMP. Those cells show minimal contraction, however; treating them with MLCK and cofactors renews contraction brought about by ATP. When DF are incubated with trifluoperazine to block calmodulin-dependent enzyme reactions, cell contraction is inhibited. Adding cytochalasin B disrupts microfilaments and also inhibits contraction. This work supports the idea that myosin ATPase is critical to cell contraction. Myosin ATPase is dependent on the phosphorylation of the regulatory peptide, myosin light chain. Elevating intracellular concentrations of cAMP or treatment of permeabilized cell preparations with alkaline phosphatase may inhibit myosin ATPase activity. The restoration of phosphorylation by adding MLCK with cofactors served to reestablish cell contraction.

Hydrophobic polyoxins are resistant to intracellular degradation in Candida albicans.

Two novel polyoxins, N-epsilon-(octanoyl)-lysyl-uracil polyoxin C (Oct-Lys-UPOC) and N-gamma-(octyl)-glutaminyluracil polyoxin C (Oct-Gln-UPOC), were synthesized by reacting uracil polyoxin C with the appropriate amino acid p-nitrophenyl ester. Oct-Lys-UPOC and Oct-Gln-UPOC were strong inhibitors (Kis = 1.7 X 10(-6)M) of chitin synthetase from Candida albicans membrane preparations. In a permeabilized-cell assay, Oct-Gln-UPOC had a 10-fold-lower inhibitory activity toward chitin synthetase than did the Oct-Lys-UPOC analog. Both compounds were resistant to hydrolysis by a cell extract of C. albicans H317; however, Oct-Gln-UPOC was hydrolyzed with a half-life of 23 min by a permeabilized-cell preparation. Oct-Lys-UPOC was resistant to hydrolysis by permeabilized cells. Oct-Gln-UPOC and Oct-Lys-UPOC did not compete with the transport of peptides or uridine into the cell. At concentrations up to 2 mM these two new polyoxins were ineffective in the inhibition of cell growth or reduction of cell viability, but they induced aberrant morphologies in C. albicans at a concentration of 0.25 mM. These data suggest that polyoxins containing hydrophobic amino acids retain strong chitin synthetase inhibitory activity and are resistant to cellular hydrolysis. They provide the first example of effective synthetic chitin synthetase inhibitors which are stable inside C. albicans.

A simple micromethod for rapid kinetic analyses of yeast enzymes in situ

A simple approach for rapid assay of enzymes in large numbers of samples of whole cells of yeast is described. The technique involves treatment of yeast cells with toluene dissolved in ethanol (1:4, v/v; TE), rendering them selectively permeable to small molecules, while letting the enzymes remain intracellular and catalytically active but accessible to exogenously added assay reagents. The permeated cell preparations permit rapid assay of nitrate reductase activity in situ [P. V. Choudary and G. Ramananda Rao (1976)Current Sci.45, 324–327]. Investigation of enzyme kinetic parameters such as pH optima, temperature dependence, and substrate saturation, which involves several samples and needs to be investigated very rapidly and accurately, is greatly facilitated by permeated cells prepared from a very small culture of cells, eliminating the need for elaborate preparations of cell extracts. By using permeabilized cell preparations, the need to supplement the reaction mixture with expensive cofactors can also be obviated to a large extent. Further, the enzyme activity displays a broader range of tolerance in situ to variations in reaction conditions compared to that in vitro. Permeabilized cell preparations thus seem to present the enzymes in the form of relatively less disintegrated functional complexes, making them a valuable tool in biochemical and cell biological investigations, and facilitate the correlation of in vitro data with the in vivo phenomena more confidently.

Recovery of sealed H35 hepatoma cells in monolayer culture following lysolecithin permeabilization

A procedure for the quantitative recovery of permeabilized H35 hepatoma cells using lysolecithin is reported. More than 80% of the cells of confluent cultures can be made permeable, which can then seal in defined medium over the next 24 h with essentially complete recovery. Dividing cultures are more susceptible to lysolecithin than confluent cultures. By reducing the concentration of lysolecithin and time of exposure, permeabilized cell preparations can be generated from dividing cultures that will seal when incubated in defined medium supplemented with serum and folinic acid.

The Effects of Cyanide and Azide on the Photoreduction of 3-Phosphoglycerate and Oxaloacetate by Wild Type and Two Reductive Pentose Phosphate Cycle Mutants of Chlamydomonas reinhardtii

3-Phosphoglycerate- and oxaloacetate-dependent O(2) photoevolution by permeabilized cell preparations (Pressates), prepared from wild type (Wt) and two reductive pentose phosphate cycle mutants of Chlamydomonas reinhardtii showed different sensitivities to the inhibitors sodium cyanide and sodium azide. NaCN (1.5 millimolar) severely inhibits both CO(2)- and 3-phosphoglycerate-dependent O(2) photoevolution by the Wt Pressate, but does not inhibit 3-phosphoglycerate-dependent O(2) photoevolution by Pressates prepared from the mutants rcl-u-1-10-6C (which lacks ribulose, 1-5, bisphosphate carboxylase activity) and F60 (which lacks phosphoribulokinase activity). NaN(3) (0.5 millimolar) inhibits 3-phosphoglycerate-dependent O(2) photoevolution by the rcl-u-1-10-6C Pressate more severely than in the Pressates prepared from F60 and Wt. A higher concentration of NaN(3) (2.0 millimolar) severely inhibited oxaloacetate-dependent O(2) photoevolution by the rcl-u-1-10-6C, but not by the F60 Pressate. O(2) exchange-dependent upon methyl viologen was not strongly inhibited by 2 millimolar NaN(3) in either of the mutant Pressates. The data suggests that the mutational lesions which resulted in decreased ribulose-1,5-bisphosphate carboxylase and phosphoribulokinase activities effected changes in other photosynthetic reactions, either by direct interactions between component proteins or by causing changes in substrate or cofactor availability to the partial reactions.

Allosteric regulation of the state of adenylylation of glutamine synthetase in permeabilized cell preparations of Escherichia coli. Studies of monocyclic and bicyclic interconvertible enzyme cascades, in situ.

Allosteric regulation of the state of adenylylation of glutamine synthetase in permeabilized cell preparations of Escherichia coli. Studies of monocyclic and bicyclic interconvertible enzyme cascades, in situ.