A new approach for assessing cumulative lysosomal degradation of proteins or other macromolecules.

A general method is proposed for the direct estimation of the degradation in various tissues of macromolecules that are metabolized by a lysosomal mechanism. This involves coupling to the macromolecule a small molecule that is cleaved from it only after entry into the lysosome, that is not metabolized but is “trapped” in the lysosome, and that therefore accumulates as a direct function of the amount of macromolecule degraded. The feasibility of the method was shown using low density lipoprotein and serum albumin doubly labeled with covalently bound [¹⁴C]sucrose and ¹²⁵I. Uptake by normal fibroblasts, measured in terms of ¹⁴C accumulated in the cells, correlated very closely with uptake measured in terms of ¹²⁵I-labeled metabolites in the medium plus ¹²⁵I in the cells.     

Culture conditions affecting induction and release of lymphocyte produced proliferation inhibitory factor (PIF)

Studies on the factors affecting the production of a proliferation inhibitory factor (PIF) by human lymphocytes are presented. Maximal PIF production occurred with mitogen stimulation of blood lymphocytes cultured at 1 × 10⁶/ml. Optimal cultures contained 10% fetal calf serum, but PIF could be produced in the absence of serum, and after only a 6-hr pulse exposure to PHA. PIF production was found to correlate with lymphocyte activation in response to the mitogen PHA but was not related to lymphocyte proliferation (DNA synthesis). Inhibitory activity could be detected as early as 3 hr after mitogen addition, long before DNA synthesis occurs. The mitogens Con A and PWM initiated different intensities of DNA synthesis in these cultures, but similar quantities of PIF. Antigenic stimulation of sensitive human peripheral lymphocyte populations resulted in the release of PIF. Cells from donors that gave a strong positive skin test to tuberculin (PPD) responded in tissue culture to PPD by producing PIF, while the cells from skin test negative donors did not. A small quantity of PIF was also evident in the supernatants from cultures with no known stimulus (“unstimulated”), this was found to result from activation of the lymphocytes by nonlymphoid elements and by fetal calf serum. An investigation of the PIF-producing capabilities of other lymphoid tissues showed that lymph node cells produced this humoral factor, whereas thymus cells did not. Thymus cell supernatants, in fact, were found to contain an extremely labile cytotoxin which degraded rapidly upon storage.     

The effects of plasma from guinea pigs with tumor on PHA stimulated lymphocyte cultures.

Strain 13 guinea pigs with a transplanted, 3-methylcholanthrene induced fibrosarcoma produced a plasma factor that inhibited PHA induced blastogenesis of lymphocyte cultures from normal and tumor animals. The suppressive activity was not cytotoxic, was not adsorbed from plasma by lymph node cells, and was a noncompetitive inhibitor of PHA activation. The tumor plasma could not inhibit the uptake of [³H]thymidine if added during the period of maximal DNA synthesis, which suggested that the suppressive factor acts most effectively on metabolically resting cells. This nonspecific suppressive factor may represent an important mechanism for allowing rapidly progressive tumors to escape immunological destruction.     

A comparison of serum-free media for the support of in vitro mitogen-induced blastogenic expansion of cytolytic lymphocytes

The high cost and potential dangers of including human AB serum in incubation media used to expand lymphocyte populations in vitro for adoptive immunotherapy have stimulated efforts to develop defined media which can support both the expansion and induction of lymphocytes with tumor cytolytic activity in the absence of serum. Lymphocyte proliferation following exposure to either PHA or the combination of phorbol 12,13-dibutyrate (PDBu) and the calcium ionophore, ionomycin, was evaluated. Although the media tested, X-Vivo 10, HB-104, AIM V, and HL-1, supported the generation of comparable levels of LAK activity after 3–5 days incubation with 10³ U human recombinant interleukin-2 (rIL-2)/ml, there were striking differences in the ability of each medium to support mitogenically stimulated lymphocytes in the absence of serum, with cells in AIM V and X-Vivo 10 showing the highest levels of DNA synthesis. In long-term cultures (17 days) of blood MNC stimulated by PDBu and ionomycin, X-Vivo 10 and HB-104 yielded the greatest numbers of cells. The addition of 2% AB serum greatly enhanced the ability of each medium to support cell proliferation to equivalent maximum levels. The results indicate that while all four serum-free media were suitable for lymphocyte culture and support the development of LAK activity, they differ in their capacity to support expansion of lymphocyte populations in response to polyclonal mitogenic activation. This latter characteristic should be considered before choosing a particular serum-free formulation as its constituents may affect mechanistic interpretations regarding signal transduction events.     

Serum factors affecting the incorporation of [3H]uridine by lymphocytes stimulated by concanavalin A. Studies of the role of complement

1. Rat lymph-node cells cultured in serum and medium 199 were activated to transform and proliferate by concanavalin A. Initial cell activation was assessed by measuring the enhanced radioactive labelling of cells with [(3)H]uridine produced by concanavalin A during the first 6h of culture. 2. In medium containing serum the degree of activation was dependent on the ratio of concanavalin A to non-diffusible serum macromolecules; however, cells could be activated to a normal extent in a medium containing only diffusible molecules. This indicates that certain serum macromolecules buffer cell receptor sites against reaction with concanavalin A. 3. At high concanavalin A concentrations labelling was depressed below that of control cultures without concanavalin A. This inhibition of labelling (i) occurred in calf serum, but not in homologous serum, (ii) was removed by pretreatment of serum with various complement inhibitors, and (iii) first appeared after 1h of culture following an initial phase of cell activation by concanavalin A. Cells pre-labelled with [(3)H]uridine slowly released the label into the culture medium; this rate of release was suddenly accelerated after 1h of culture with concanavalin A if complement was present. The results suggest that inhibition of labelling requires the sequential binding of concanavalin A and then complement to the cell surface. 4. Results from experiments in which calf serum was mixed in various proportions with calf serum which had been preheated to inactivate complement, suggest (i) a requirement for complement in stoicheiometric quantities dependent on the number of cells being inhibited, and (ii) that preheated serum can inactivate complement in unheated serum. 5. The proliferative response over 3 days of culture was assessed by measuring the enhanced labelling of cells with [(3)H]thymidine produced by concanavalin A. In preheated calf serum two types of inhibition were noted. (i) A progressive inhibition at high concanavalin A concentrations so that the optimum response was shifted to lower concanavalin A concentrations as the duration of culture was extended; it is suggested that this reflects the secretion of complement by cultured cells. (ii) An inhibition of the optimum response appearing late in the culture period at high cell concentrations; it is suggested that this is due to the exhaustion of medium nutrients in most actively growing cultures.     

A method for observing protein-protein interaction.

A method is proposed for observation of the interaction between charged macromolecules such as proteins. The method is based on the fact that the pK of an ionizable reporter group attached to a macromolecule can be altered by the electrostatic effect of another charged macromolecule which associates with the former. The effectiveness of the method was shown in the study of the association of bovine serum albumin with hen egg lysozyme [EC 3.2.1.17]. The errors inherent in this method in obtaining the equilibrium constant of the association reaction and procedures for their correction are discussed.     

Effects of mitogens on sodium-potassium transport, H-ouabain binding, and adenosine triphosphatase activity in lymphocytes

The effect of various mitogens was studied on sodium (Na⁺) potassium (K⁺) transport, ³H-ouabain binding, and adenosine triphosphatase (ATPase) activity in human and sheep peripheral lymphocytes. Concanavalin A (ConA), phytohemagglutinin (PHA), horse anti-lymphocyte serum (ALS), and anti-IgG antisera, in order of decreasing potency, stimulated in particular the ouabain-sensitive K⁺ pump influx, while the cardiac glycoside-insensitive K⁺ leak flux was only slightly affected. Sheep lymphocytes primed in vivo with human IgG as antigen also responded with K⁺ pump flux activation when exposed to the antigen in vitro. Both PHA and ConA also stimulated active Na⁺ efflux in human lymphocytes. Apparently these mitogens activate the Na⁺K⁺ pump system in the lymphocyte membrane—an assumption supported by the finding of a significant activation of the ouabain-sensitive Na⁺K⁺-ATPase. From rate studies of ³H-ouabain binding carried out at 37 °C in presence and absence of sodium azide, and at 0 °C, it is concluded that PHA alters the rate of ouabain uptake to these cells. Thus PHA may alter the affinity of the pump for ouabain, equivalent to an increased cation turnover per pump site. However, our findings do not completely discount the possibility that PHA also increases the total number of ouabain molecules bound and therefore of Na⁺K⁺ pumps.     

Folate requirement for blast cell transformation in mixed leukocyte cultures.

Mixed leukocyte cultures from normal donors were set up in standard tissue culture media. Blast cell transformation was measured 6 days later by ³H-thymidine uptake and assessed qualitatively with stained smears. Media RPMI 1629 and RPMI 1640 allowed much more intense blastogenesis than Medium 199, the difference being due to the low folic acid content of Medium 199 (0.01 mg/liter) compared with RPMI 1629 (10 mg/liter) and RPMI 1640 (1 mg/liter). Further experiments indicated that folic acid increased the multiplication rate of blast cells but did not promote the induction or initial transformation phases of the mixed leukocyte reaction. The effect of folic acid on the PHA response was entirely different: ³H-thymidine uptake in 3 day PHA cultures was decreased, and there was no apparent effect on the number or percentage of blast cells seen on the smears. The reason for this difference is at present unknown, but some possibilities are discussed.     

Rapidly metabolized glycoproteins in a neuroplastoma cell line

The metabolism of neuroblastoma cell glycoproteins was examined using l-[³H]fucose. Incubation of monolayer cultures with [³H]fucose resulted in a rapid uptake of the radioactive precursor and its incorporation into acid-insoluble macromolecules. Less than 3% of the [³H]fucose that was isolated from neuroblastoma cells by trichloroacetic acid precipitation was associated with glycolipids. The metabolism of fucosylated macromolecules was studied in cells which were labelled to a steady state, and then reincubated under conditions which limited reutilization of the radioactive precursor (40 mM unlabelled fucose). During reincubation of the cells, we observed a rapid metabolism (27% by 2 h)_ of the prelabelled macromolecules which stabilized within a cell generation time to give an overall rate of turnover of 9%. This rapid loss of radioactivity from the cells was not due to exocytosis since less than 4% of the [³H]-fucose was lost into the media as macromolecules during a 5 h reincubation period. The presence of 40 mM fucose in the media did not affect cell growth until after 24 h of incubation or cellular synthesis until after 15 h of incubation. When the metabolism of neuroblastoma cell glycoproteins was measured in the presence of 1.8 · 10^?4 M cycloheximide, there appeared to be a less rapid decrease in cell-associated specific activity, and an increased reutilization of [³H]fucose. Although the major proportion of the radioactivity remained as [³H]fucose, extensive incubation of neuroblastoma cells with this radioactive precursor led to increased amounts of tritium associated with other cellular components. However, a rapid rate of glycoprotein metabolism could also be demonstrated with cells incubated with [⁴C]fucose. This eliminated the possibility that the above results were restricted to the tritiated precursor and merely a reflection of hydrogen-tritium exchange.     

Methylation of macromolecules during development in Myxococcus xanthus.

Covalent modification of macromolecules can serve to alter their biological activities and is therefore frequently involved in regulation. I examined methylation of proteins and carbohydrates during development and vegetative growth in the procaryote Myxococcus xanthus. Striking differences in the patterns of protein methylation occurred when cell development was induced by nutrient deprivation on solid media and when cells were starved in liquid. In addition, a methylated, protease-resistant macromolecule which contained carbohydrate and which may have been an unusual type of lipopolysaccharide was observed on sodium dodecyl sulfate-polyacrylamide gels. A comparison of methylation patterns in various media and an analysis of the time course of methylation indicated that changes in methylation were part of the developmental pathway which includes aggregation. Induction of development in liquid by glycerol produced no changes in methylation.     

Immune function in aged mice. I. T-cell responsiveness using phytohaemagglutinin as a functional probe.

The loss of cell-mediated immunity with age was assessed by a detailed analysis of the in vitro response of murine lymphocytes to the well-defined probe of T-cell function, PHA (phytohaemagglutinin). The reduced mitogenic activity of lymphoid cells from old mice compared with young mice could not be explained in terms of a shift in kinetics of the responding cells. Removal of macrophages, which are known to exert a regulatory effect on T-cell function, failed to reverse the poor response of old lymphoid cells. Furthermore, no evidence was found for a role of soluble inhibitors released by either lymphocytes or macrophages in the decreased response of old cells. Not only were old cells less efficient in producing such factors, but in addition, they responded less well to them than did young cells. Taken together, these observations implied that the defect in PHA responsiveness of old cells is due to a disturbance in the T cells themselves rather than to any extracellular influences. The total number of T cells, assessed by labelling with anti-Thy-1 serum was comparable in old and young animals. Selective depletion of a subpopulation of PHA-reactive cells was excluded by direct quantitation of PHA-binding cells. Thus, 25% of small lymphocytes from the spleens of old mice bound ¹²⁵I-labelled PHA ([¹²⁵I]PHA) compared with 15% in the case of young mice. To show that the cells binding PHA were those reacting to it, a suicide technique was used. Spleen cells pretreated with [¹²⁵I]PHA failed to respond to subsequent challenge with the specific mitogen, but could mount a normal response to a control (B-cell), mitogen, LPS (lipopolysaccharide). When PHA cultures were carried out in the presence of colchicine, fewer cells from old mice were found to react to the mitogenic signal. In the absence of evidence for depletion of precursor cells, the conclusion was reached that the T-cell defect in old mice is more likely to be qualitative than quantitative, perhaps due to metabolic or structural abnormalities preventing lymphocyte transformation and/or proliferation.     

The macromolecule with antimicrobial activity synthesized by <Emphasis Type="Italic">Pseudoalteromonas luteoviolacea</Emphasis> strains is an <Emphasis Type="SmallCaps">l</Emphasis>-amino acid oxidase

Two purple pigmented bacterial strains, CPMOR-1 and CPMOR-2, have been newly isolated from the Mediterranean Sea. 16S RNA sequencing and phenotypic characteristics indicate that they belong to the species Pseudoalteromonas luteoviolacea. The synthesis of macromolecules with antimicrobial activity is a capacity described in many strains of this species although the nature of those macromolecules has not been reported up to now. The search for antimicrobial compounds in the two new strains described in this work shows that they synthesize a macromolecule with antimicrobial activity that can be inhibited by catalase, as it had been described in the type strain P. luteoviolacea NCIMB 1893(T). This work elucidates the nature of such macromolecule as a novel L-amino acid oxidase (LAO) with broad substrate specificity. The enzyme is most active with Met, Gln, Leu, Phe, Glu, and Trp. In growth media containing those amino acids, the hydrogen peroxide generated by the reaction catalyzed by the LAO mediates its antimicrobial activity.     

Sub-mitogenic phorbol myristate acetate co-stimulation rescues the PHA-induced activation of both naïve and memory T cells cultured in the rotating-wall vessel bioreactor

T lymphocytes are unresponsive to T cell receptor (TCR) stimulation during culture in spaceflight or ground-based microgravity analogs such as the rotating-wall vessel (RWV) bioreactor. The TCR-induced activation of a subset of T cells can be rescued in the RWV by co-stimulation with sub-mitogenic doses of phorbol ester (PMA). We report that PMA co-stimulation of primary human T cells cultured in the RWV rescues the phytohemagglutinin (PHA)-induced activation of the CD8⁺ and CD4⁺ T cell subsets as well as naïve and memory CD4⁺ T cells. Importantly, T cells activated in the RWV by PHA + PMA contained these subsets in proportions strikingly similar to control cultures activated with PHA alone. The data indicate that rescuing T cell activation with PMA co-stimulation does not significantly perturb the heterogeneity of the responding cells, and represent an important proof of principle for the design of immune-boosting agents for use in spaceflight.     

Characterization of medium-chain-length polyhydroxyalkanoate biosynthesis by Pseudomonas mosselii TO7 using crude glycerol

Polyhydroxyalkanoates (PHAs) are biopolyesters produced by microorganisms that are environmentally friendly. PHAs can be used to replace traditional plastic to reduce environmental pollution in various fields. PHA production costs are high because PHA must be produced from a carbon substrate. The purpose of this study was to find the strain that can used the BDF by-product as the sole carbon source to produce high amounts of medium-chain-length PHA. Three isolates were evaluated for potential PHA production by using biodiesel-derived crude glycerol as the sole carbon source. Among them, Pseudomonas mosselii TO7 yielded high PHA content. The PHA produced from P. mosselii TO7 were medium-chain-length-PHAs. The PHA content of 48% cell dry weight in 48 h with a maximum PHA productivity of 13.16 mg PHAs L^?1 h^?1. The narrow polydispersity index value of 1.3 reflected the homogeneity of the polymer chain, which was conducive to industrial applications.     

The Golgi apparatus mediates the transport of phytohemagglutinin to the protein bodies in bean cotyledons

When developing cotyledons of Phaseolus vulgaris L. were labeled with [³H]fucose, fucose-labeled phytohemagglutinin (PHA) was found in organelles with average densities of 1.13 g cm^-3 and 1.22 g cm^-3. The position of these organelles on isopycnic sucrose gradients was independent of the presence of MgCl₂ and ethylenediaminetetraacetate in the media, indicating that the fucose-labeled PHA was not associated with the rough endoplasmic reticulum (ER). The organelles with a density of 1.13 g cm^-3 were identified as membranes of the Golgi apparatus on the basis of the similarity of their sedimentation properties and those of the Golgi marker enzyme, inosine diphosphatase, in both isopycnic and rate-zonal sucrose gradients. The organelles with a density of 1.22 g cm^-3 were identified as small (0.1–0.4 μm), electron-dense vesicles with a protein content similar to that of the protein bodies. Pulsechase experiments with [³H]fucose indicated that fucose-labeled PHA first appeared in the Golgi-apparatus-derived membranes and later in the dense vesicles. Fucose-labeled PHA chased out of the Golgi apparatus first, then out of the dense vesicles, and accumulated in the soluble portion of the homogenate which contained the contents of the broken protein bodies. Fucose-labeled PHA chased out of the two types of organelles with a t _1/2 of 20–30 min, a rate three to four times faster than newly synthesized PHA chases out of the bulk of the ER (Chrispeels, M.J., Bollini, R., 1982, Plant Physiol. 70, 1425–1428). This result indicates that the Golgi apparatus is a much smaller compartment than the ER in the storage parenchyma cells. The sodium ionophore, monensin, which interferes with the function of the Golgi apparatus of animal cells, blocks the biosynthesis and—or transport of fucose- and galactose-labeled macromolecules to the cotyledon cell walls. Monensin also blocks the transport of labeled PHA out of the Golgi apparatus and into the protein bodies. These results provide the first biochemical evidence that a specific storage protein which accumulates in seeds is modified in, and passes through, the Golgi apparatus on its way to the protein bodies.     

Ecto-protein kinase activity of fibroblasts.

Isolated, intact dermal fibroblasts can transfer the terminal phosphate of adenosine triphosphate, [γ-³²P]ATP, to an exogenously added macromolecule (histone). The incorporation of labeled phosphate to histone is attributed to an extracellularly directed protein kinase activity (ecto-kinase) which cannot be accounted for by soluble cytoplasmic protein kinase that might have been released and become bound to cell membranes during the cell preparation. The addition of soluble cytoplasmic enzyme preparations to the cell suspension was fully recoverable in the supernatant and the first wash. The activity of ectokinase was abolished by incubation of intact cells with trypsin for 5 min, whereas the activity of cytoplasmic enzyme was unaffected by the trypsin treatment. These data suggest that dermal fibroblasts contain protein kinase on the outer surface of plasma membrane which can phosphorylate exogenously added macromolecules. The ecto-protein kinase activity is dependent on cell number, time of incubation, and the concentration of Mg²⁺ in the reaction mixture. Lineweaver-Burk plot analyses yielded K_m values for ATP and histone of 7 × 10^?5 and 3 × 10^?6m, respectively. The ecto-protein kinase activity of normal fibroblasts and fibrosarcoma cells were also compared. The enzyme activity of normal cells was higher than that of the malignant cells and was not significantly affected by cyclic nucleotides, whereas the activity of the malignant cells were stimulated by the addition of micromolar concentrations of the cyclic nucleotides.     

Incorporation of fucose into the carbohydrate moiety of phytohemagglutinin in developing Phaseolus vulgaris cotyledons

Incubation of developing cotyledons of P. vulgaris with [³H]fucose resulted in the incorporation of radioactivity into the cell wall, membranous organelles and soluble macromolecules. Fractionation of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by fluorography, showed that phytohemagglutinin (PHA) was the major fucosylated protein synthesized in the cotyledons. Incorporation of fucose into PHA occurred in the membranous organelle fraction, and the radioactive fucose remained associated with the PHA during a 20-h chase of the radioactivity. Tunicamycin inhibited the incorporation of glucosamine and fucose into PHA to the same extent (65%), indicating the involvement of a lipid intermediate in the incorporation of fucose, or the attachment of fucose to the high-mannose oligosaccharide moiety of newly synthesized PHA. Digestion with proteinase K of [³H]fucose- or [³H]glucosamine-labeled PHA resulted in the formation of glycopeptides of similar size. These glycopeptides were partially resistant to digestion with endo-β-N-acetylglucosaminidase H, even after the removal of fucose by mild acid hydrolysis. We postulate, on the basis of these experiments, that the transport of PHA from the endoplasmic reticulum to the protein bodies is accompanied by the modification of its oligosaccharide side-chain. This modification involves inter alia the attachment of fucose, and renders the oligosaccharide side-chain resistant to digestion with endo-β-N-acetylglucosaminidase H. Analogy with animal glycoproteins indicates that this modification probably occurs in the Golgi apparatus.     

Human chorionic gonadotropin beta-core fragment is directly produced by cancer cells

The present study was undertaken to investigate whether hCG beta-core fragment (hCGbeta cf) was directly produced by cancer cells. Fifteen cell lines, including four choriocarcinoma and five ovarian cancer cell lines, were tested, and immunoreactivity of hCGbeta cf was present in the culture media of five of the cell lines. It was also present in the culture media of Chinese hamster ovary (CHO) cells transfected with hCGbeta gene. In addition to hCGbeta cf, gel chromatography and Western blot analysis of the culture media showed the presence of an hCGbeta cf immunoreactive material with a molecular weight of approximately 40 kDa. In an in vivo study, hCGbeta cf immunoreactivity was detected in the sera of the mice transplanted with NaUCC-3 choriocarcinoma cells, although the ratios of hCGbeta cf/hCG and hCGbeta cf/free hCGbeta were lower than those in the culture medium. Incubation experiments of purified hCGbeta cf in the serum showed no substantial decrease in its values, ruling out the possibility that formation of a macromolecule with serum components may mask hCGbeta cf immunoreactivity in the serum. Taken together, these results indicate that hCGbeta cf immunoreactive materials are directly produced by cancer cells and hCGbeta cf is not a urinary metabolite of hCG or hCGbeta alone. Also, reduced levels of hCGbeta cf in the serum compared with that of intact hCG or free hCGbeta are likely due to its short half-life.     

ESR investigation of the binding of acidic biopolymers to synthetic apatite.

The interaction between synthetic crystalline calcium phosphate (apatite) and acidic macromolecules (sodium polyacrylate, sodium poly(L -glutamate), chondroitin sulfate, phosvitin) was investigated by electron spin resonance spectroscopy of mineral-macromolecule complexes doped with vanadyl ion (VO⁺⁺) as a paramagnetic probe. Changes in magnetic parameters were interpreted in terms of bonding between mineral and macromolecule. The VO⁺⁺ probe data indicated that polymer acidic functional groups were bound to mineral surfaces in all cases.     

Quantification of Intracellular Polyhydroxyalkanoates by Virtue of Personalized Flow Cytometry Protocol

Polyhydroxyalkanoates (PHAs) are natural polyesters produced by microbes, a potential alternative to synthetic plastics. Various methods ranging from gravimetry to spectrophotometry are routinely used for qualitative analysis of extracted PHA. There is a great need for accurate quantification of intracellular PHA during bioprocess. Hence, the present study aims to improvise the existing Nile red-based flow cytometry protocol. It was achieved using respective cells in a non-PHA accumulating state as gating control to minimize non-specific staining. The optimal Nile red concentration required for PHA staining is 5?×?10³?pg?mL^?1, which is ~10³-fold less than that of earlier reports. Further, it was inferred that flow-based quantification was more accurate than the gravimetric method. The intracellular PHA content was highest in Pseudomonas sp. MNNG-S (52.06?%) among the Pseudomonas strains tested by the flow-based method. Both gravimetric and flow-based cell cycle analyses revealed that DNA synthesis (S phase) and PHA production (log phase) are synchronous at 24–48?h of culture. This study supports flow-based PHA quantification for real time online measurement of intracellular PHA for bioreactor monitoring, control and optimization enduing industrial applications.