dimitrova

K e y w o r d s : grehlin signaling, hormone, orexigenic, prostaglaudin, smooth muscle, tromboxane Furthermore, hormones and local mediators often change the conductivity of ion channels in the cell membrane, which can be used as sensors for proper signaling. Our pilot study showed that ghrelin reduces the iberiotoxin-sensitive Ca 2+ -activated potassium current (I K(Ca) ) elicited in freshly isolated smooth muscle cells of human mesenteric arteries via PLD-and PKCdependent mechanism (15). The sarcoplasmic reticulum is also necessary for this signaling as the blockade of sarco-endoplasmic reticulum Ca 2+ ATPase or IP 3 -activated Ca 2+ channels of internal Ca 2+ stores inhibit the effect of ghrelin on I K(Ca) In the present study, we used pharmacological tools to identify the participants of ghrelin signaling and found a second mediator involved. MATERIALS AND METHODS The investigation conformed to the 'Declaration of Helsinki' 1975Helsinki' (revised 1983. Mesenteric arteries were isolated from extracted specimens of human mesentery taken during abdominal surgery on patients -63 men aged 64.6±1.5 years and 43 women aged 59.3±1.9 -and transported to the laboratory in ice-cold saline. Half of the patients were operated for malignant growths (carcinoma sigma) and the rest -for nonmalignant conditions. Contraction studies Segments of mesenteric arteries were dissected, carefully cleaned of adipose and connective tissues and kept in ice-cold low Ca 2+ solution containing (mmol): 118 NaCl, 5 KCl, 1.2 MgCl 2 , 0.16 CaCl 2 , 10 glucose, 1.2 Na 2 HPO 4 and 24 HEPES. Arterial rings (2 mm long) were mounted on a wire-myograph for isometric tension recording DMT, model 410A (Danish Myo Technology, Aarhus, Denmark) whose chamber was filled with the same ice-cold low Ca 2+ solution. After the mounting of the vessel rings, the organ bath solution was replaced with the same solution containing 2.5 mmol CaCl 2 . The bath was heated up to 37°C and continually bubbled with carbogen (95% O 2 and 5% CO 2 ). The isometric force of contraction was recorded using the program Myodaq (DMT, Aarhus, Denmark). The arterial segments were equilibrated for 1 hour at 37°C in a buffer, which was changed at least 3 times during this equilibration period. In most experiments, the endothelium was removed by careful rubbing with a rat whisker. Then vessels were stretched to their optimal lumen diameter, corresponding to 90% of the passive diameter of the vessel at 100 mm Hg. The viability of the preparations was tested twice by application of 10 µmol noradrenaline. The integrity of the endothelium was tested with 10 µmol acetylcholine added to 10 µmol noradrenaline contracted rings. After the viability tests, the strips were contracted with 1 nmol ET-1, which produced relatively stable isometric contractions allowing the study of the effect of the increasing concentrations of ghrelin. The tension reached a steady state in about 40 minutes after the application of ET-1. Then ghrelin was applied to the bath in increasing concentrations of 10, 30, 100, 300 and 1000 nmol, i.e. starting from a value that is about 10 times higher than its plasma level. It led to a significant effect on native artery preparations (with endothelium and in the absence of TTX) at a 30-100 times higher concentration than in human circulation (11). A possible explanation of this result is that ghrelin has a low diffusion rate through the adventitia, which decreases its interaction with receptors of smooth muscle cells when applied to the bath solution. Other researchers have also used higher ghrelin concentrations while studying the effect of ghrelin on vascular preparations (12), probably due to the same reason. It is also possible that ghrelin reaches such values in the smooth muscle layer of the arterial wall due to its paracrine release from human vascular endothelium (16). Additionally, guinea pigs may have a higher plasma level of ghrelin if compared to humans or rats. The ghrelin-induced changes in tension were expressed as a percentage of the maximum tension elicited by 1 nmol ET-1. The influence of different pharmacological agents (inhibitors) on ghrelin effect was studied by means of their addition to the bath about 40 min after ET-1 application and incubated for about 30 min before the application of ghrelin. The effects of inhibitors were studied using several types in vitro preparations: i) native (untreated) preparations of small human mesenteric arteries; ii) endothelium-denuded preparations; iii) native preparations with tetrodotoxin (TTX, 300 nmol) and mainly iv) endotheliumdenuded and TTX-treated preparations. The time control preparations were equally treated, but instead of ghrelin, an equal volume of solvent (deionised water) was added at the same time intervals. The inhibitors and antagonist were applied to block (to switch off) the studied enzyme or receptor activity and not to induce a partial inhibition only. This forced us to use a higher concentration of these substances. On the other hand, the possibility of a non-specific effect of the pharmacological tools restricted us to using them in lower concentrations. Thus, the aim to choose the optimal concentration of each substance for our experiments was not easy. For almost all of the blockers, however, there are at least several studies on arterial preparations in vitro, in some cases with dose-response curves and/or tests for cross-reaction. Besides, the choice of each concentration was based on our earlier experience with a significant part of the substances used either in electrophysiological or functional studies of different vascular beds. Whole-cell patch-clamp experiments This method has been described in detail elsewhere (17). In brief, whole-cell voltage-clamp experiments were performed on single smooth muscle cells, freshly isolated from human mesenteric arteries. The arteries were cut into 3 mm long pieces and placed in 0.1 mmol Ca 2+ -containing physiological salt solution (PSS, for composition see below) warmed to 37°C and containing 1.5 mg ml -1 collagenase II, 1 mg ml -1 papain, 15 µl ml -1 elastase and 1 mg ml -1 albumin. After 30 to 35 min incubation at 37°C with continuous O 2 bubbling, the enzymes were washed away and the tissue pieces triturated 5 times in Ca 2+ -free PSS using a pipette with a small tip opening. The remainder of the tissue was put back into the enzyme-containing solution for another 5 min and then carefully washed with Ca 2+ -free PSS. Single smooth muscle cells were obtained by gentle trituration in 2 ml of the same Ca 2+ -free solution. Cells could be stored for up to 8 hours in this solution at 4-6°C. The external solution (PSS) for single-cell voltage experiments contained (in mmol): 126 NaCl, 5.6 KCl, 10 HEPES, 20 taurine, 20 glucose, 1.1 MgCl 2 , 0.8 CaCl 2 , 5 Napyruvate and pH was adjusted to 7.4 with NaOH. The same solution was used for the isolation of cells. The solutions in the recording pipette contained (in mmol): 125 KCl, 6 NaCl, 10 HEPES, 1 MgCl 2 , 3 EGTA, 0.1 ATP, 5 Na-pyruvate, 5 succinate, 5 oxalacetate, 5 glucose and 2.15 CaCl 2 to give a calculated free Ca 2+ of 200 nmol and pH was adjusted to 7.4 with KOH. Chemicals Most of the substances used for solution preparation were obtained from ICN (Irvine, CA, USA). NaOH, BSA, 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC), pertussis toxin, indomethacin, O-(octahydro-4,7-methano-1H-inden-5-yl) carbonopotassium dithioate (D-609), collagenase type II, prostaglandine F 2α (PGF 2α ), (5Z,13E) -(9S,11S,15R)-384 9,15,dihydroxy-11-fluoro-15-(2-indanyl)- Data analysis Current densities were expressed in pA/pF and plotted as functions of the potential applied to obtain data suitable for statistical analysis. The significance of differences between means was assessed using Tukey-Kramer multiple comparison test with p<0.05 regarded significant. The force of contraction was evaluated as a difference in tension (N/m) measured before ET-1 application and the plateau reached afterwards. The contractile effect of ghrelin was expressed as a percentage of the maximal ET-1 induced contraction, taken as 100%. Values were expressed as means±S.E.M. From five to ten human mesenteric arterial preparations (n) were included in the construction of each concentration-response curve of ghrelin. Data were subjected to a comparative statistical analysis one-way ANOVA with Bonferroni correction (p<0.05). RESULTS Octanoyl ghrelin, herein referred to as ghrelin, dosedependently increased the force of contraction of isometric human mesenteric artery preparations constricted with ET-1 The application of increasing doses of ghrelin to external solution containing either NCDC (50 mol) ( The application of increasing concentrations of ghrelin to TTX-and ET-1-containing bath solution failed to influence significantly the force of contraction of endothelium-denuded human mesenteric arteries in the presence of PP2 (10 µmol) -a selective Src family kinase inhibitor The addition of ghrelin (100 nmol) almost entirely inhibited the iberiotoxin-sensitive I K(Ca) recorded during a 500 ms depolarizing pulse to +40 mV from a holding potential of -50 mV (12). Rp-cAMPS (200 µmol), a specific membranepermeable inhibitor of PKA, was without effect on the total outward potassium current (I K ) (n=5), while the subsequent addition of ghrelin (100 nmol) decreased I K to the same degree as in the absence of this PKA inhibitor in single smooth muscle cells from human mesenteric arteries (n=5; p<0.01) DISCUSSION Ghrelin and des-octanoyl ghrelin are equipotent antagonists of ET-1 induced vasoconstriction of human mammary artery (13) while in single smooth muscle cells isolated from human mesenteric arteries des-octanoyl ghrelin blocks the ghrelininduced inhibition of I K(Ca) . This difference supposes the operation of more than one ghrelin receptors in human vascular beds -the des-octanoyl ghrelin-blockable GHS-R1a in human mesenteric arteries and another type in human mammary artery (for a review of ghrelin receptors see 19). Kleinz et al. (13) routinely applied indomethacin to exclude the possibility of endothelium dependent vasodilatation. This treatment however blocks not only endothelial COX1/2 but also those in the tunica media of the artery and thus eliminates the influence of COX1/2 downstream products generated in smooth muscle cells, as suggested by our study. Indeed, such a mechanism is unexpected in blood vessels but is reported in non-vascular smooth muscle (lower esophageal sphincter) that maintains mainly tonic type of contraction similarly to arteries (20). Therefore, it is still difficult to summarize the mechanisms of ghrelin effects on human arteries due to their opposite influences -relaxation and contraction, the different experimental conditions applied and the need for more detailed studies of the intracellular participants. Most often GHS-R1a interacts with heterotrimeric G q/11 proteins and stimulates the G q/11 /PI-PLC/IP 3 +Ca 2+ +DAG/PKC signaling (2). It was reported that the application of a specific PKC inhibitor entirely abolished the effect of ghrelin on I K(Ca) , recorded in single smooth muscle cells of human mesenteric arteries (15). In our study the force of contraction of human mesenteric arteries did not respond to ghrelin application if iberiotoxin or GF109203x were present in the bath solution. Thus, ghrelin requires activation of PKC and suppresses K Ca channels with a large conductance (BK Ca channels) to increase the force of contraction. Ghrelin regulates cAMP/PKA (3-5) and cGMP/PKG signaling (6), which can further influence ion channels. Han et al. (6) reported a ghrelin-induced reduction of voltage-gated I K of rat anterior pituitary tumor (GH3) cells by a PKG-dependent mechanism and Kohno et al. (21) -a ghrelin-induced activation of N-type Ca 2+ channels that required PKA. BK Ca channels are involved in the formation of the spontaneous artery tone, counteract the elevation of the agonist-induced cytosolic free Ca 2+ and participate in the relaxation induced by cAMP/PKAand cGMP/PKG-coupled agonists (22). Therefore, we investigated the participation of both cyclic nucleotides using specific inhibitors Rp-cAMPS for PKA and ODQ for soluble guanylate cyclase. The presence of inhibitors of PKA or soluble guanylate cyclase in the bath solution did not prevent the effect of ghrelin on I K(Ca) . We concluded that the second messengers cAMP and cGMP are not involved in the observed ghrelininduced inhibition of I K(Ca) . On the other hand, in human mesenteric arteries the effect of ghrelin on the force of contraction is blocked by pertussis toxin, which suggests the participation of G i -proteins. In rat islet β-cells ghrelin decreases the insulin secretion by a G αi2 -protein sensitive activation of voltage-gated K + channels and this effect is blocked by D-Lys 3 -GHRP-6, a specific GHS-R1a inhibitor (23). Ghrelin inhibits BK Ca channels in the guinea pig femoral artery via a pertussis toxin and GHS-R1a sensitive pathway (17). Ghrelin also activates G i -protein in cell cultures Ca 2+ released from sarcoplasmic reticulum activates BK Ca channels of smooth muscles cells (24). In human mesenteric arteries IP 3 -sensitive Ca 2+ release is essential for the ghrelininduced decrease of I K(Ca) (15) and for the increase of the force of contraction. Additionally, IP 3 -induced Ca 2+ release from sarcoplasmic reticulum participates in the ET-1 evoked contraction of this vascular bed. Our pharmacological studies suggest that several DAG-producing phospholipases (PI-PLC and PC-PLC) are important in establishing the effect of ghrelin in human mesenteric arteries. We presume that PI-PLC is essential mainly for triggering the contraction (25) and for the IP 3 -induced Ca 2+ release-dependent translocation of PKC to the plasma membrane, while the sustained DAG producer PC-PLC (25) is responsible for the long lasting PKC activation. This suggestion is indirectly supported by the slowly developing inhibition (in 10-14 min) of I K(Ca) by ghrelin in this tissue (15). Using different inhibitors we reveal several new enzymes participating in the ghrelin effect in human mesenteric arteries. Thus, the selective inhibition of MEK or Src kinase entirely blocks the ghrelin-induced contractions in endothelium-denuded human mesenteric arteries with suppressed neurotransmission. The nonselective COX1/2 inhibitor indomethacin eliminates either the ghrelin-induced constriction of human mesenteric arteries or the ghrelin-induced decrease of I K in single smooth muscle cells isolated from the same tissue. All these data suggest the existence of a ghrelin-induced and pertussis toxin-sensitive mechanism, which increases the force of contraction by a consequent activation of Src kinase, MEK and ERK. Similar G iprotein initiated signaling was reported for non-vascular tissues (for review see 26). Next, the stimulated ERK may activate the cytosolic PLA 2 (27), which increases vascular arachidonic acid production -the rate-limiting step for prostaglandin synthesis (28). COX1/2 transform this arachidonic acid into PGE 2 , and then PGE 2 into PGH 2 , which may further yield contracting prostaglandin or thromboxane, as reported for non-vascular smooth muscle It was reported that ghrelin receptor type GHR-R1a has the ability to oligomerize with prostanoid receptors, when they are transiently over-expressed in human embryonic kidney 293 cells (32). The same authors stated that this co-transfection significantly influenced GHR-R1a activity without changes in its affinity for ghrelin. Similarly, as an alternative explanation of our data, it is suggested that ghrelin first binds to GHS-R1a and then activates prostanoid receptor via a direct interaction in the existing GHS-R1a/prostanoid receptor heteromeric complex. If this is the case, the enzymes necessary to demonstrate the effect of ghrelin on the contractile activity (Src kinase, MEK, COX-1 and thromboxane synthase) only support the steady-state thromboxane A 2 production and are not additionally activated by ghrelin. The second explanation of our data, however, seems to be less probable as several new articles report a direct activation of ERK1/2 (3, 7, 33) and Src kinase (34) by ghrelin signaling. Ghrelin decreases the mean arterial pressure of the rat by a COX-insensitive and NOS-sensitive mechanism (35). In rat mesenteric arteries both ghrelin and desacyl ghrelin evoke endothelium-dependent dilation by NOS-and COX-insensitive mechanism (36). Ghrelin inhibits the contraction of human aortic smooth muscle cells by cAMP/PKA pathway activation (4). Ghrelin and desacyl ghrelin antagonize the ET-1-induced contraction of human internal mammary artery (13). Ghrelin decreases the mean arterial pressure in humans as well (11). On the other hand, contractile effects of ghrelin were reported in guinea pig femoral (37) and renal (17) arteries and in rat coronary artery (38). Ghrelin increases the force of contraction of human mesenteric arteries partially constricted with ET-1. The effect is stronger in endothelium-denuded preparations and most pronounced in endothelium-denuded artery segments with blocked action potential propagation of perivascular neurons. These data point to a relaxing effect of ghrelin via endothelium and axonal projections in adventitia, which antagonize the direct and stronger contractile action of ghrelin on the smooth muscle layer of the vascular wall. Additionally, if compared to native preparations during the first half of the experiments, the higher force of contractions of endothelium-denuded human mesenteric arteries suggest that endothelium, as well as perivascular neurotransmission are functional, i.e. they are not badly damaged by the therapy before the surgical intervention or during the transportation. It can be concluded that ghrelin either increases or decreases the force of contraction of arteries depending on their type and the species. Thus, ghrelin and desacyl ghrelin may influence the artery resistance similarly to other regulators of the circulation with opposite effects on different vascular beds. For example, catecholamines redistribute the blood flow throughout the body, depending on the physiological needs, via different adrenergic receptors and intracellular mechanisms. In summary, ghrelin has been shown to increase the force of contraction of human mesenteric arteries by a novel mechanism that requires active Src kinase, MEK, COX-1 and thromboxane synthase and that depends on the release of a local mediator -a T prostanoid receptor agonist. Additionally, our data suggest a novel physiological regulation, in which an empty stomachinitiated increase of ghrelin secretion reduces the abdominal circulation in adult humans until next meal. Acknowledgement

A topologically twisted index for three-dimensional supersymmetric theories

We provide a general formula for the partition function of three-dimensional (formula presented) gauge theories placed on S2 7S1 with a topological twist along S2, which can be interpreted as an index for chiral states of the theories immersed in background magnetic fields. The result is expressed as a sum over magnetic fluxes of the residues of a meromorphic form which is a function of the scalar zero-modes. The partition function depends on a collection of background magnetic fluxes and fugacities for the global symmetries. We illustrate our formula in many examples of 3d Yang-Mills-Chern-Simons theories with matter, including Aharony and Giveon-Kutasov dualities. Finally, our formula generalizes to \u3a9-backgrounds, as well as two-dimensional theories on S2 and four-dimensional theories on S2 7 T2. In particular this provides an alternative way to compute genus-zero A-model topological amplitudes and Gromov-Witten invariants

Effective Rheology of Bubbles Moving in a Capillary Tube

We calculate the average volumetric flux versus pressure drop of bubbles moving in a single capillary tube with varying diameter, finding a square-root relation from mapping the flow equations onto that of a driven overdamped pendulum. The calculation is based on a derivation of the equation of motion of a bubble train from considering the capillary forces and the entropy production associated with the viscous flow. We also calculate the configurational probability of the positions of the bubbles.Comment: 4 pages, 1 figur

Endothelial Progenitor Cell Number and Colony-forming Capacity in Overweight and Obese Adults

OBJECTIVE: To investigate whether adiposity influences endothelial progenitor cell (EPC) number and colony-forming capacity.DESIGN: Cross-sectional study of normal weight, overweight and obese adult humans.PARTICIPANTS: Sixty-seven sedentary adults (aged 45-65 years): 25 normal weight (body mass index (BMI) or=30 kg/m(2); 18 males/6 females). All participants were non-smokers and free of overt cardiometabolic disease.MEASUREMENTS: Peripheral blood samples were collected and circulating EPC number was assessed by flow cytometry. Putative EPCs were defined as CD45(-)/CD34(+)/VEGFR-2(+)/CD133(+) or CD45(-)/CD34(+) cells. EPC colony-forming capacity was measured in vitro using a colony-forming unit (CFU) assay.RESULTS: Number of circulating putative EPCs (either CD45(-)/CD34(+)/VEGFR-2(+)/CD133(+) or CD45(-)/CD34(+) cells) was lower (P\u3c0.05) in obese (0.0007±0.0001%; 0.050±0.006%) compared with overweight (0.0016±0.0004%; 0.089±0.019%) and normal weight (0.0015±0.0003%; 0.082±0.008%) adults. There were no differences in EPC number between the overweight and normal weight groups. EPC colony-formation was significantly less in the obese (6±1) and overweight (4±1) compared with normal weight (9±2) adults.CONCLUSION: These results indicate that: (1) the number of circulating EPCs is lower in obese compared with overweight and normal weight adults; and (2) EPC colony-forming capacity is blunted in overweight and obese adults compared with normal weight adults. Impairments in EPC number and function may contribute to adiposity-related cardiovascular risk

Pre-Procedural Atorvastatin Mobilizes Endothelial Progenitor Cells: Clues to the Salutary Effects of Statins on Healing of Stented Human Arteries

OBJECTIVES: Recent clinical trials suggest an LDL-independent superiority of intensive statin therapy in reducing target vessel revascularization and peri-procedural myocardial infarctions in patients who undergo percutaneous coronary interventions (PCI). While animal studies demonstrate that statins mobilize endothelial progenitor cells (EPCs) which can enhance arterial repair and attenuate neointimal formation, the precise explanation for the clinical PCI benefits of high dose statin therapy remain elusive. Thus we serially assessed patients undergoing PCI to test the hypothesis that high dose Atorvastatin therapy initiated prior to PCI mobilizes EPCs that may be capable of enhancing arterial repair. METHODS AND RESULTS: Statin naïve male patients undergoing angiography for stent placement were randomized to standard therapy without Atorvastatin (n = 10) or treatment with Atorvastatin 80 mg (n = 10) beginning three days prior to stent implantation. EPCs were defined by flow cytometry (e.g., surface marker profile of CD45dim/34+/133+/117+). As well, we also enumerated cultured angiogenic cells (CACs) by standard in vitro culture assay. While EPC levels did not fluctuate over time for the patients free of Atorvastatin, there was a 3.5-fold increase in EPC levels with high dose Atorvastatin beginning within 3 days of the first dose (and immediately pre-PCI) which persisted at 4 and 24 hours post-PCI (p<0.05). There was a similar rise in CAC levels as assessed by in vitro culture. CACs cultured in the presence of Atorvastatin failed to show augmented survival or VEGF secretion but displayed a 2-fold increase in adhesion to stent struts (p<0.05). CONCLUSIONS: High dose Atorvastatin therapy pre-PCI improves EPC number and CAC number and function in humans which may in part explain the benefit in clinical outcomes seen in patients undergoing coronary interventions

Impairment of circulating endothelial progenitors in Down syndrome

<p>Abstract</p> <p>Background</p> <p>Pathological angiogenesis represents a critical issue in the progression of many diseases. Down syndrome is postulated to be a systemic anti-angiogenesis disease model, possibly due to increased expression of anti-angiogenic regulators on chromosome 21. The aim of our study was to elucidate some features of circulating endothelial progenitor cells in the context of this syndrome.</p> <p>Methods</p> <p>Circulating endothelial progenitors of Down syndrome affected individuals were isolated, <it>in vitro </it>cultured and analyzed by confocal and transmission electron microscopy. ELISA was performed to measure SDF-1α plasma levels in Down syndrome and euploid individuals. Moreover, qRT-PCR was used to quantify expression levels of <it>CXCL12 </it>gene and of its receptor in progenitor cells. The functional impairment of Down progenitors was evaluated through their susceptibility to hydroperoxide-induced oxidative stress with BODIPY assay and the major vulnerability to the infection with human pathogens. The differential expression of crucial genes in Down progenitor cells was evaluated by microarray analysis.</p> <p>Results</p> <p>We detected a marked decrease of progenitors' number in young Down individuals compared to euploid, cell size increase and some major detrimental morphological changes. Moreover, Down syndrome patients also exhibited decreased SDF-1α plasma levels and their progenitors had a reduced expression of SDF-1α encoding gene and of its membrane receptor. We further demonstrated that their progenitor cells are more susceptible to hydroperoxide-induced oxidative stress and infection with Bartonella henselae. Further, we observed that most of the differentially expressed genes belong to angiogenesis, immune response and inflammation pathways, and that infected progenitors with trisomy 21 have a more pronounced perturbation of immune response genes than infected euploid cells.</p> <p>Conclusions</p> <p>Our data provide evidences for a reduced number and altered morphology of endothelial progenitor cells in Down syndrome, also showing the higher susceptibility to oxidative stress and to pathogen infection compared to euploid cells, thereby confirming the angiogenesis and immune response deficit observed in Down syndrome individuals.</p

Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles

Release of membrane vesicles, a process conserved in both prokaryotes and eukaryotes, represents an evolutionary link, and suggests essential functions of a dynamic extracellular vesicular compartment (including exosomes, microparticles or microvesicles and apoptotic bodies). Compelling evidence supports the significance of this compartment in a broad range of physiological and pathological processes. However, classification of membrane vesicles, protocols of their isolation and detection, molecular details of vesicular release, clearance and biological functions are still under intense investigation. Here, we give a comprehensive overview of extracellular vesicles. After discussing the technical pitfalls and potential artifacts of the rapidly emerging field, we compare results from meta-analyses of published proteomic studies on membrane vesicles. We also summarize clinical implications of membrane vesicles. Lessons from this compartment challenge current paradigms concerning the mechanisms of intercellular communication and immune regulation. Furthermore, its clinical implementation may open new perspectives in translational medicine both in diagnostics and therapy