Both monovalent cations and plectin are potent modulators of mechanical properties of keratin K8/K18 networks.

Intermediate filament (IF) networks are a major contributor to cell rigidity and thus serve as vital elements to preserve the integrity of entire cell layers. Keratin K8 and K18 IFs are the basic constituents of the cytoskeleton of epithelial cells. The mechanical properties of K8/K18 networks depend on the structural arrangements of individual filaments within the network. This paper investigates the architecture of these networks in vitro under the influence of the monovalent cation potassium and that of the cytolinker protein plectin. Whereas increasing amounts of potassium ions lead to filament bundling, plectin interlinks filaments at filament intersection points but does not lead to bundle formation. The mechanics of the resulting networks are investigated by microrheology with assembled K8/K18 networks. It is shown that bundling induced by potassium ions significantly stiffens the network. Furthermore, our measurements reveal an increase in plectin-mediated keratin network rigidity as soon as an amount corresponding to more than 20% of the plectin present in cells is added to the keratin IF networks. In parallel, we investigated the influence of plectin on cell rigidity in detergent-extracted epithelial vulva carcinoma derived A431 cells in situ. These cytoskeletons, containing mostly IFs, actin filaments and associated proteins, exhibit a significantly decreased stiffness, when plectin is downregulated to ≈10% of the normal value. Therefore, we assume that plectin, via the formation of IF-IF connections and crosslinking of IFs to actin filaments, is an important contributor to cell stiffness.

Strong dipole-quadrupole coupling and Fano resonance in H-like metallic nanostructures.

Under certain conditions of the incident light polarization direction a Fano resonance arises in small gold nanorods arranged in a H-like configuration. This stems from the coupling between a bright dipole plasmon mode excited in the horizontal rod and dark quadrupole plasmon modes in both vertical rods. We investigate these surface plasmon modes, and analyze the dependence of the Fano resonance on the geometry parameters such as rod size and interparticle separation, and refractive index of embedding medium. To describe the degree of this energy transfer, we introduce a new parameter: the Fano resonance efficiency. We calculate absorption cross-sections for visible and NIR spectrum in each element of the structure, and near-field distributions at different wavelengths. We show that Fano resonance in small H-like structures exhibits high sensitivity with respect to the refractive index of the host medium, outperforming the values for larger plasmonic structures based on nanorods already investigated.

Microrheology of keratin networks in cancer cells.

Microrheology is a valuable tool to determine viscoelastic properties of polymer networks. For this purpose measurements with embedded tracer beads inside the extracted network of pancreatic cancer cells were performed. Observing the beads motion with a CCD-high-speed-camera leads to the dynamic shear modulus. The complex shear modulus is divided into real and imaginary parts which give insight into the mechanical properties of the cell. The dependency on the distance of the embedded beads to the rim of the nucleus shows a tendency for a decreasing storage modulus. We draw conclusions on the network topology of the keratin network types based on the mechanical behavior.

Safety of nurse-directed triage intranasal fentanyl protocol for acute pain management in a European pediatric emergency department: A retrospective observational analysis.

Background: Nurse-directed pain protocols for intranasal fentanyl administration are not widely implemented in European (EU) pediatric emergency departments (PED). Barriers include perceived safety concerns for intranasal (IN) fentanyl. The aim of this study is to describe our experience with a nurse-directed triage IN fentanyl protocol with a focus on safety in a tertiary EU PED. Methods: We conducted a retrospective analysis of patient records of children aged 0-16 years who received nurse-directed IN fentanyl between January 2019 and December 2021 at the PED of the University Children's Hospital of Bern, Switzerland. Extracted data points included demographics, presenting complaint, pain score, IN fentanyl dosage, concomitant pain medication use, and adverse events. Results: A total of 314 patients were identified with ages ranging from 9 months to 15 years. The main indication for nurse-directed fentanyl administration was musculoskeletal pain due to trauma (n = 284, 90%). Mild adverse events (vertigo) were reported in two patients (0.6%), without a correlation to concomitant pain medication or protocol violation. The only reported severe adverse event of syncope and hypoxia in a 14-year-old adolescent occurred in a setting where the institutional nurse-directed protocol was violated. Conclusion: In accordance with previous studies outside of Europe, our data support the case that when appropriately used, nurse-directed IN fentanyl is a safe potent opioid analgesic for pediatric acute pain management. We strongly encourage the introduction of nurse-directed triage fentanyl protocols Europe-wide in order to provide effective and adequate acute pain management in children.

The scanning ion-conductance microscope.

A scanning ion-conductance microscope (SICM) has been developed that can image the topography of nonconducting surfaces that are covered with electrolytes. The probe of the SICM is an electrolyte-filled micropipette. The flow of ions through the opening of the pipette is blocked at short distances between the probe and the surface, thus, limiting the ion conductance. A feedback mechanism can be used to maintain a given conductance and in turn determine the distance to the surface. The SICM can also sample and image the local ion currents above the surfaces. To illustrate its potential for imaging ion currents through channels in membranes, a topographic image of a membrane filter with 0.80-micrometer pores and an image of the ion currents flowing through such pores are presented.

Scanning tunneling microscopy and atomic force microscopy: application to biology and technology.

The scanning tunneling microscope (STM) and the atomic force microscope (AFM) are scanning probe microscopes capable of resolving surface detail down to the atomic level. The potential of these microscopes for revealing subtle details of structure is illustrated by atomic resolution images including graphite, an organic conductor, an insulating layered compound, and individual adsorbed oxygen atoms on a semiconductor. Application of the STM for imaging biological materials directly has been hampered by the poor electron conductivity of most biological samples. The use of thin conductive metal coatings and replicas has made it possible to image some biological samples, as indicated by recently obtained images of a recA-DNA complex, a phospholipid bilayer, and an enzyme crystal. The potential of the AFM, which does not require a conductive sample, is shown with molecular resolution images of a nonconducting organic monolayer and an amino acid crystal that reveals individual methyl groups on the ends of the amino acids. Applications of these new microscopes to technology are demonstrated with images of an optical disk stamper, a diffraction grating, a thin-film magnetic recording head, and a diamond cutting tool. The STM has even been used to improve the quality of diffraction gratings and magnetic recording heads.

Atomic force microscopy of an organic monolayer.

Atomic force microscope images of polymerized monolayers of n-(2-aminoethyl)-10,12-tricosadiynamide revealed parallel rows of molecules with a side-by-side spacing of approximately equal to 0.5 nanometer. Forces used for imaging (10(-8) newton) had no observable effect on the polymer strands. These results demonstrate that atomic force microscope images can be obtained for an organic system.

Influence of the light-scattering form factor on the Bragg diffraction patterns of arrays of metallic nanoparticles.

Accurate models for the light-scattering form factors of nanoparticles are of crucial importance to characterize the optical properties of the particles and to develop new photonic devices. Analytical or semi-empirical models exist for particles of spherical and cylindrical shape. The angular spectrum of scattering for particles of more complex shape is very complex and can only be obtained by numerical simulations. Moreover, the light scattering of metallic particles depends on many parameters as size, shape, optical constants, substrate and polarization of light. Experimental verification of the differential scattering cross-sections obtained from different calculation methods is always necessary. Measurements done on single nanoparticles are very sensitive to their local properties and the signal-to-noise ratio may be very poor. Arrays of identical particles illuminated by a planes waves produce Bragg diffraction and the resultant patterns depend on the averaged values of the form factors of the particles. In order to test the validity of models for the scattering form factor, we present an experimental setup capable of measuring Bragg diffraction patterns of arrays of nanoparticles in the visible and near-infrared regions of the spectrum. The approach is similar to that of X-ray diffraction of crystals.

A single lipopolysaccharide administration is sufficient to induce a long-term desensitization of the hypothalamic-pituitary-adrenal axis.

We have previously shown that a single exposure of adult rats to a severe emotional stressor such as immobilization is able to exert a long-term desensitization of the response of the hypothalamic-pituitary-adrenal (HPA) axis to the same stimulus when applied days to weeks later. Surprisingly, the intensity of the effect increased with time elapsed between the two exposures, suggesting that we are dealing with a new type of stress-associated phenomenon. Taking into account the clinical importance of tolerance to endotoxin, in the present study we assessed whether a single exposure to an immunological stressor such as lipopolysaccharide can induce effects similar to those of immobilization. Rats injected with lipopolysaccharide (1 mg/kg) showed a reduction of the response of the corticotropin-releasing factor mRNA in the paraventricular nucleus of the hypothalamus after a new lipopolysaccharide injection 4, but not 2 weeks later. In an additional experiment using a different blood sampling procedure, adrenocorticotropin hormone, corticosterone and tumor necrosis factor-alpha responses were reduced approximately to the same extent by previous experience with lipopolysaccharide either 1 or 4 weeks before. Our data suggest that a previous single exposure to lipopolysaccharide induces a long-lasting tolerance of the HPA axis that likely involves some kind of learning-like brain plasticity.

Acute stress attenuates but does not abolish circadian rhythmicity of serum thyrotrophin and growth hormone in the rat.

The effects of acute immobilization (IMO) on daily rhythms of corticosterone, thyroid-stimulating hormone (TSH) and growth hormone (GH) were studied in adult male rats. Two hours of IMO increased serum corticosterone, this increase still being observed 3 h after finishing stress exposure. In the dark period corticosterone levels did not differ in control and IMO rats, but higher levels were observed again in the morning of the day after. Immobilization lowered serum GH and TSH levels throughout the 24-h period that followed exposure to the stressor. Such an effect was more marked in GH than in TSH. In addition, GH, but not TSH, levels were found to be reduced significantly by IMO at 08.30 h of the next day. None the less, daily rhythms of GH and TSH were still persistent and roughly similar to those of control rats. The daily rhythm of food intake was measured in a separate experiment and it was observed, as expected, that IMO reduced food intake only in the dark period of the lighting cycle. It appears therefore unlikely that IMO-induced anorexia was the major factor responsible for the inhibition of GH and TSH caused by IMO at 11.00 and 19.00 h, considering that the amount of food intake was very low and similar in control and IMO rats during this period. However, anorexia might have contributed to inhibition of GH and TSH secretion afterwards. Thus, in a third experiment we studied the contribution of IMO-induced anorexia to the changes in hormone levels observed 24 h after stress by introducing a group of pair-fed rats. It was found that IMO, but not pair-feeding, reduced TSH levels, whereas a similar reduction of GH was found in the two conditions. It might be concluded that acute stress transiently altered corticosterone secretion, the only long-lasting effect being a slight increase in its morning levels on the following stress. Immobilization also causes an inhibition of GH and TSH secretion in the rat that persists for several hours after finalization of exposure to the stressor, but daily rhythms were still apparent. It appears that the contribution of stress-induced anorexia is different in GH than in TSH. In conclusion, an acute severe stressor such as IMO, although modifying circulating levels of some hormones, particularly in the hours following exposure to the stressor, did not appear to interfere greatly with the expression of circadian rhythms of anterior pituitary hormones.

Effect of regularity of exposure to chronic immobilization stress on the circadian pattern of pituitary adrenal hormones, growth hormone, and thyroid stimulating hormone in the adult male rat.

Circadian variation of serum levels of adrenocorticotropin hormone (ACTH), corticosterone, growth hormone (GH), and thyroid-stimulating hormone (TSH) were studied in three groups of adult male rats exposed to chronic intermittent immobilization stress (IMO) for 2 hr daily under different schedules. IMO resulted in reduced food intake, body weight loss, and increased adrenal weight. ACTH levels were not affected but corticosterone levels were increased in all IMO rats as compared to control ones during the diurnal phase of the circadian cycle. IMO decreased serum GH and TSH levels but the circadian pattern of secretion was influenced in a complex way depending on the specific pattern of daily exposure to IMO. Differences observed between the IMO groups were not caused by differences in food intake because its circadian rhythm was very similar in all IMO groups. These results suggest that regularity of exposure to immobilization alters in a complex fashion circadian GH and TSH rhythms.

Acute stress markers in humans: response of plasma glucose, cortisol and prolactin to two examinations differing in the anxiety they provoke.

Although the endocrine response to psychological stressors has been extensively studied both in animals and humans, the sensitivity of these variables to the intensity of stress experienced by exposure to psychologically stressful situations has not been studied in humans. In the present work this was assessed by measuring plasma levels of glucose, cortisol and prolactin in female medical students just before taking two examinations clearly differing in the anxiety they provoke. It was found that both examinations increased anxiety just before taking them, but the physiology examination (EPh) caused higher anxiety than the psychology (EPs). Prolactin increased in response to both examinations as compared to the non-stress condition, but its levels were greater in the EPh than in the EPs. Cortisol followed the same pattern as prolactin, but increased only marginally in the EPs. Finally, glycemia rose to the same extent in response to both examinations. A significant positive correlation was found between anxiety and glucose, and between cortisol and prolactin when data from all situations were included. On the basis of these results, it appears that the three variables might be useful as putative markers of stress in humans, although glucose might reflect different underlying psychological processes than cortisol and prolactin. In addition, it was found for the first time that prolactin is able to discriminate between stressful situations of different intensity. The response of these physiological variables to other stressful situations differing both in quantitative and in qualitative terms merits to be studied in further work.

Effects of chronic immobilization stress on GH and TSH secretion in the rat: response to hypothalamic regulatory factors.

The effect of chronic immobilization (2 h/day) for 13 days on basal and stress levels of GH and TSH, and their response to various hypothalamic regulatory factors was studied in male Sprague-Dawley rats. Chronic immobilization (IMO) resulted in reduced serum TSH levels in stress situations but not in resting conditions. GH secretion was inhibited both in resting and stress situations. Chronic IMO impaired both GH and TSH responses to GRH and TRH, respectively, but also to another peptide (VIP) stimulatory for the two hormones. Whereas somatostatin administration inhibited GH secretion in control but not in chronic IMO rats, its inhibitory effect on TSH was slight and similar in the two experimental groups. The present results suggest that chronic exposure to a severe stressor such as IMO alters GH and TSH secretion, at least in part by changes in the response of the pituitary to the hypothalamic regulatory factors. The actual influence of chronic IMO on the release of these peptides into the median eminence remains to be studied.

Long-term effects of a single exposure to immobilization on the hypothalamic-pituitary-adrenal axis: neurobiologic mechanisms.

In apparent contrast to previous results from other labs, we have found that a single exposure to a severe stressor such as immobilization (IMO) caused a long-term desensitization of the hypothalamic-pituitary-adrenal (HPA) response to the homotypic stressor. Because such HPA desensitization was not found in response to heterotypic stressors, it seemed at first that we were describing a habituation process already observed after a single experience with the stressor. However, a more detailed analysis revealed two main properties incompatible with the interpretation of the results in terms of habituation: (1) The intensity of desensitization increases over the course of days to weeks with no additional exposures to the stressor, and (2) the degree of desensitization was greater with more severe stressors. The long-term effects were also observed after a single exposure to a high dose of a systemic stressor such as endotoxin but not after insulin-induced hypoglycemia, suggesting that not all severe systemic stressors can induce such long-term desensitization. Because systemic stressors are known to be processed in specific brain areas and because we have found changes in c-fos mRNA response to the homotypic stressor in some brain areas as a consequence of previous experience with IMO, we hypothesize that some severe stressors do not induce long-term desensitization because they are not processed in brain areas sensitive to previous experience with the stressor. The neurochemical mechanisms involved in the induction of long-term effects on the HPA axis are in process, but our results suggest only a partial role of glucocorticoids and NMDA receptors.

The hypothalamic-pituitary-adrenal axis in autoimmunity.

We have characterized the activation of the HPA axis in the chronic inflammatory stress model of adjuvant-induced arthritis. Alteration in the hypothalamic control mechanism, where CRF is no longer the major corticotrophin-releasing factor, has been noted in a number of other immune-mediated disease models, including experimental allergic encephalomyelitis, eosinophilia myalgia syndrome, systemic lupus erythematosus, and leishmaniasis. These changes occur in both the mouse and the rat, suggesting this may be a common mechanism to chronic immune activation. We have good evidence to suggest that AVP takes over as the major stimulator of the axis. The arthritic rat is unable to mount a response to acute stressors, such as restraint or ip hypertonic saline. However, these animals are able to mount a response to an acute immune challenge. These data provide further evidence for a differential activation of the HPA by acute stress or acute immune stimulation. This presumably reflects an adaptive response to the development of chronic inflammation. We have demonstrated that central neurotransmitter systems are able to influence the severity of peripheral inflammation. In particular we have shown that depletion of serotonin at the time of the development of the inflammatory episode reduces the severity of the inflammation. These findings suggest the possibility of novel therapeutic strategies targeting neurotransmitter systems to alleviate inflammation.

Novel ligands for the investigation of imidazoline receptors and their binding proteins.

New ligands for imidazoline receptors are described so that these receptors can be more fully explored and understood. BU224, (2-(4,5-dihydroimidaz-2-yl)-quinoline, shows high affinity and is selective for the imidazoline-2 (I(2)) class of receptors. BU224 was tested in the rat Porsolt forced swim paradigm where it was found to decrease time spent immobile and increase the time spent swimming, consistent with an antidepressant profile. BU224 was tritiated and, in radioligand binding studies, was found to label a single population of saturable sites with high affinity. In vitro brain autoradiography with [(3)H]BU224 also showed a pattern of distribution similar to the known labeling of I(2) receptors. A new series of four 2BFI (2-(benzofuranyl)-2-imidazoline) derivatives were investigated as potential ligands for imaging brain I(2) receptors using positron emission tomography (PET). At least two, BU20012 and BU20013, retained high affinity and moderate selectivity and penetrated the brain when administered peripherally in the mouse. 2BFI has undergone the Mannich reaction to immobilized diaminodipropyl amine to fabricate an affinity column, which was used to isolate a protein from rabbit brain; this protein was sequenced and identified as the enzyme creatine kinase.

Emergent immunoregulatory properties of combined glucocorticoid and anti-glucocorticoid steroids in a model of tuberculosis.

In Balb/c mice with pulmonary tuberculosis, there is a switch from a protective Th1-dominated cytokine profile to a non-protective profile with a Th2 component. This switch occurs while the adrenals are undergoing marked hyperplasia. Treatment with the anti-glucocorticoid hormones dehydroepiandrosterone or 3 beta, 17 beta-androstenediol, during the period of adrenal hyperplasia, maintains Th1 dominance and is protective. We investigated the effects of these hormones as therapeutic agents by administering them from day 60, when the switch to the non-protective cytokine profile was already well established. Given at this time (day 60), doses that were protective when given early (from day 0) were rapidly fatal. A physiological dose of the glucocorticoid corticosterone was also rapidly fatal. However when the corticosterone and the anti-glucocorticoid (AED or DHEA) were co-administered, there was protection, with restoration of a Th1-dominated cytokine profile, enhanced DTH responses, and enhanced expression of IL-1 alpha and TNF alpha. Therefore this combination of steroids has an emergent property that is quite unlike that of either type of steroid given alone. It may be possible to exploit the ant-inflammatory properties of glucocorticoids while preserving a Th1 bias, by combining glucocorticoids with DHEA or suitable metabolites.

Influence of Regularity of Exposure to Chronic Stress on the Pattern of Habituation of Pituitary-Adrenal Hormones, Prolactin and Glucose.

The effect of regularity of exposure to two different chronic stressors (noise or immobilization (IMO)) on the pattern of habituation of pituitary-adrenal (PA) hormones, prolactin and glucose was evaluated in adult male rats. Animals were chronically subjected to either regular or irregular time schedule of noise (30 min/day) or IMO (2 h/day) for two weeks. The day after the last stress session the rats were killed without stress or after having been subjected to 30 min of the homotypic stressor. Whereas regular noise did not affect food intake, body weight gain or adrenal weight, irregular noise decreased body weight gain and induced a moderate adrenal hypertrophy. In addition, previous daily exposure to regular but not to irregular noise reduced both prolactin and corticosterone responses to acute noise. In contrast, glucose response to acute noise was reduced after both regular and irregular exposure to chronic noise. Either regular or irregular exposure to chronic IMO decreased food intake and body weight and increased adrenal weight to the same extent. Likewise, no influence of regularity of exposure to chronic IMO on corticosterone and prolactin responses to acute IMO was observed. However, habituation of the ACTH response to acute IMO was observed in rats subjected to chronic regular IMO, but not in rats subjected to chronic irregular IMO. Finally, acute IMO-induced hyperglycemia diminished to the same extent after regular and irregular IMO. From these results we can conclude that: first, the process of habituation of the PA axis to chronic stress is greatly dependent upon factors such as regularity of exposure to the stressor and stressor intensity, and second, the influence of regularity on the pattern of habituation to a repeated stressor is dependent on the physiological variable we are dealing with.

Influence of single or repeated experience of rats with forced swimming on behavioural and physiological responses to the stressor.

In the present work behavioural (struggling and immobility), physiological (hypothermia, glycaemia) and endocrine (hypothalamo-pituitary-adrenal (HPA) hormones) response to repeated forced swimming (FS) for 15 days was studied in adult male rats and compared with the response of rats having only one single experience with FS either 1 or 14 days before the last exposure to the stressor. Repeated experiences with FS reduced struggling and increased immobility as compared with stress-naïve rats, whereas a single previous exposure to FS, regardless of the time elapsed, had the same, but less marked, effect. Hypothermia followed the same trend. FS-induced hyperglycaemia was not sensitive to a previous single experience, but rather it was totally abolished in chronically stressed rats. Neither a single nor chronic exposure to FS modified the secretion of ACTH in response to the last FS session. However, repeated FS enhanced the speed of recovery of plasma corticosterone as compared to control rats, suggesting a dissociation between the two hormones. The present results revealed great differences in the sensitivity of various behavioural and physiological responses to repeated FS stress and suggest that reduced response to repeated FS, when found, is not a consequence of the time elapsed between exposures but to the repetition of the stressful situation.

Activation of the hypothalamic-pituitary axis in adrenalectomised rats: potentiation by chronic stress.

The influence of chronic stress on the status of the hypothalamo-pituitary-adrenal (HPA) axis of sham-operated and adrenalectomised rats was assessed. Animals underwent bilateral adrenalectomy (ADX) and 3 days later they were either left undisturbed or subjected daily to immobilization for 2 h each morning for 14 days (chronic IMO). In situ hybridization histochemistry revealed that ADX increased corticotropin-releasing factor (CRF) mRNA levels in the paraventricular nucleus of the hypothalamus (PVN) and proopiomelanocortin (POMC) mRNA levels in the anterior pituitary, in both control and chronically stressed rats as measured on the day following the last exposure to stress. Chronic IMO increased CRF mRNA levels in the PVN and POMC mRNA levels in the anterior pituitary of sham-operated rats, as measured on the day following the last exposure to stress. Chronic IMO potentiated the increase in CRF mRNA in the PVN following ADX and resulted in further increases in CRF mRNA above levels seen in adrenal-intact animals. Finally, chronic stress, while not altering basal ACTH levels of ADX rats, reduced the ACTH response of these animals to a novel stressor (tail-shock for 30 min). These results suggest that chronic stress exerts a stimulatory influence at the hypothalamic level that is partially restrained by daily stress-induced glucocorticoid release. Despite the potentiation by chronic stress of CRF mRNA content in the PVN of ADX rats, a blunted circulating ACTH response to an acute short-term stressor was apparent in ADX-chronically stressed rats, suggesting that chronic stress might also alter POMC processing and/or ACTH secretory patterns in the anterior pituitary in ADX animals.