Hypereosinophilia during dupilumab treatment in patients with chronic rhinosinusitis with nasal polyps.

BACKGROUND: Increased blood eosinophil count (BEC) is common in patients under dupilumab treatment for chronic rhinosinusitis with nasal polyps (CRSwNP). This study investigated the prevalence and consequences of hypereosinophilia and to help define patients at risk. METHODS: Real-life, prospective observational cohort study of patients treated with dupilumab for severe CRSwNP. Eligible patients were adult and biological-naive (N=334). All BEC values at baseline and during treatment were reported. Patients with a follow-up of >= 1 year were included to define patients at risk for hypereosinophilia by comparing baseline BEC values (N=218). Furthermore, clinical characteristics and therapeutic consequences for patients with BEC >= 3.0 were noted. RESULTS: Hypereosinophilia developed in a minority of patients, with a peak at week 12 (16.2% with BEC >= 1.5, and 1.7% >= 3.0) in cross-sectional analysis. BEC >= 1.5 developed in 28.9% and BEC >=3.0 in 4.6% of cases with a minimal 1-year follow-up. Baseline BEC was significantly higher for patients developing BEC >= 1.5 and BEC >=3.0, with an optimal cut-off point of 0.96 to predict developing BEC >= 3.0. CONCLUSIONS: Blood eosinophil count (BEC) >= 1.5 is transient and usually abates with no therapeutic interventions and BEC >= 3.0 is rare. Hypereosinophilic syndrome did not occur and switching to a different biological was rarely employed. A baseline BEC of >=1.0 can be a reason for extra caution.

Assessment of factual recall and higher-order cognitive domains in an open-book medical school examination.

Open-book examinations (OBEs) will likely become increasingly important assessment tools. We investigated how access to open-book resources affected questions testing factual recall, which might be easy to look-up, versus questions testing higher-order cognitive domains. Few studies have investigated OBEs using modern Internet resources or as summative assessments. We compared performance on an examination conducted as a traditional closed-book exam (CBE) in 2019 (N = 320) and a remote OBE with free access to Internet resources in 2020 (N = 337) due to COVID-19. This summative, end-of-year assessment focused on basic science for second-year medical students. We categorized questions by Bloom's taxonomy ('Remember', versus 'Understand/Apply'). We predicted higher performance on the OBE, driven by higher performance on 'Remember' questions. We used an item-centric analysis by using performance per item over all examinees as the outcome variable in logistic regression, with terms 'Open-Book, 'Bloom Category' and their interaction. Performance was higher on OBE questions than CBE questions (OR 2.2, 95% CI: 2.14-2.39), and higher on 'Remember' than 'Understand/Apply' questions (OR 1.13, 95% CI: 1.09-1.19). The difference in performance between 'Remember' and 'Understand/Apply' questions was greater in the OBE than the CBE ('Open-Book' * 'Bloom Category' interaction: OR 1.2, 95% CI: 1.19-1.37). Access to open-book resources had a greater effect on performance on factual recall questions than higher-order questions, though performance was higher in the OBE overall. OBE design must consider how searching for information affects performance, particularly on questions measuring different domains of knowledge.

Selective fish passage: Restoring habitat connectivity without facilitating the spread of a non-native species.

River managers are challenged to address two key threats to freshwater biodiversity. The first is the effects of habitat fragmentation by instream structures, such as dams and weirs, that disrupt migrations and impact species distributions. The second is the impact of non-native species on native species and ecological processes. However, mitigating anthropogenic habitat fragmentation through the installation of passage facilities can facilitate the invasion and spread of non-native species. This study compared the potential of two existing low-cost fish passage technologies designed for sloping weirs, a cylindrical bristle cluster (CBC) array and horizontally oriented studded tiles, to facilitate upstream movement of native European fish while preventing dispersal of non-native American signal crayfish (Pacifastacus leniusculus); thus providing a selective fish passage solution. Crayfish movement and passage was experimentally quantified at a Crump weir installed in a recirculating flume under two velocity regimes (low and high), without (control) and with the addition of either a CBC array or studded tiles. Results were compared to passage efficacy (PE) data for native fish species for both technologies (existing data). Most (84.4%) crayfish were active during the trials, exhibiting frequent up and downstream movements below the weir. During control conditions under the high velocity regime, high velocities (ca. 2.39 m s-1) prevented crayfish reaching the foot of the weir (PE: 0%). Under the low velocity regime, relatively low velocities (ca. 0.74 m s-1) at the weir crest prevented most crayfish from passing (PE: 10-16%). Crayfish movement speed and total distance moved were lower under the high than the low velocity regime. Neither fish pass technology improved crayfish maximum distance of ascent on the downstream weir face or PE under either velocity regime. Under comparable conditions to the high velocity regime tested here, previous studies have shown both technologies improve PE for native fish. Hence, both CBC arrays and studded tiles would likely function as suitable selective fish passes where the conservation objective is not to aid the spread of non-native crayfish. Additional passage inhibiting technologies will be required at sites where complete blockage of crayfish movement is required.

Evaluation of droplet-vitrification, vacuum infiltration vitrification and encapsulation-dehydration for cryopreservation of Syzygium maire zygotic embryos.

BACKGROUND: Syzygium maire is a threatened tree species with limited information on long-term storage options for its recalcitrant seed. OBJECTIVE: To evaluate the cryopreservation of S. maire zygotic embryo axes (EA) using dehydration, encapsulation-dehydration as well as PVS2 vitrification using droplet vitrification (DV) and the novel droplet vacuum infiltration vitrification (DVIV) methods. MATERIALS AND METHODS: Excised naked and sodium alginate encapsulated EA were desiccated to various moisture contents (MC) using a laminar flow cabinet. Moisture content, embryo survival and plantlet formation, before and after cryopreservation, were assessed at 1 h intervals during the desiccation period (0-6 h). The influence of PVS2, using DV and DVIV, was assessed for various desiccation times and temperatures. RESULTS: Encapsulated EA desiccated to 31% and 37% MC survived but no plantlets formed following cryopreservation. Exposure to PVS2 using the DV method had a negative impact on embryo survival and plantlet formation, while DVIV resulted in improved results for non-cryopreserved EA. However, neither PVS2 vitrification method resulted in embryo survival following cryopreservation. CONCLUSION: S. maire embryos are sensitive to desiccation and likely require physical, chemical or a combination of protection methods to increase embryo survival and plantlet formation following cryopreservation.

The vascular Ca2+-sensing receptor regulates blood vessel tone and blood pressure.

The extracellular calcium-sensing receptor CaSR is expressed in blood vessels where its role is not completely understood. In this study, we tested the hypothesis that the CaSR expressed in vascular smooth muscle cells (VSMC) is directly involved in regulation of blood pressure and blood vessel tone. Mice with targeted CaSR gene ablation from vascular smooth muscle cells (VSMC) were generated by breeding exon 7 LoxP-CaSR mice with animals in which Cre recombinase is driven by a SM22α promoter (SM22α-Cre). Wire myography performed on Cre-negative [wild-type (WT)] and Cre-positive (SM22α)CaSR(Δflox/Δflox) [knockout (KO)] mice showed an endothelium-independent reduction in aorta and mesenteric artery contractility of KO compared with WT mice in response to KCl and to phenylephrine. Increasing extracellular calcium ion (Ca(2+)) concentrations (1-5 mM) evoked contraction in WT but only relaxation in KO aortas. Accordingly, diastolic and mean arterial blood pressures of KO animals were significantly reduced compared with WT, as measured by both tail cuff and radiotelemetry. This hypotension was mostly pronounced during the animals' active phase and was not rescued by either nitric oxide-synthase inhibition with nitro-l-arginine methyl ester or by a high-salt-supplemented diet. KO animals also exhibited cardiac remodeling, bradycardia, and reduced spontaneous activity in isolated hearts and cardiomyocyte-like cells. Our findings demonstrate a role for CaSR in the cardiovascular system and suggest that physiologically relevant changes in extracellular Ca(2+) concentrations could contribute to setting blood vessel tone levels and heart rate by directly acting on the cardiovascular CaSR.

Increased expression of GDF-15 may mediate ICU-acquired weakness by down-regulating muscle microRNAs.

RATIONALE: The molecular mechanisms underlying the muscle atrophy of intensive care unit-acquired weakness (ICUAW) are poorly understood. We hypothesised that increased circulating and muscle growth and differentiation factor-15 (GDF-15) causes atrophy in ICUAW by changing expression of key microRNAs. OBJECTIVES: To investigate GDF-15 and microRNA expression in patients with ICUAW and to elucidate possible mechanisms by which they cause muscle atrophy in vivo and in vitro. METHODS: In an observational study, 20 patients with ICUAW and seven elective surgical patients (controls) underwent rectus femoris muscle biopsy and blood sampling. mRNA and microRNA expression of target genes were examined in muscle specimens and GDF-15 protein concentration quantified in plasma. The effects of GDF-15 on C2C12 myotubes in vitro were examined. MEASUREMENTS AND MAIN RESULTS: Compared with controls, GDF-15 protein was elevated in plasma (median 7239 vs 2454 pg/mL, p=0.001) and GDF-15 mRNA in the muscle (median twofold increase p=0.006) of patients with ICUAW. The expression of microRNAs involved in muscle homeostasis was significantly lower in the muscle of patients with ICUAW. GDF-15 treatment of C2C12 myotubes significantly elevated expression of muscle atrophy-related genes and down-regulated the expression of muscle microRNAs. miR-181a suppressed transforming growth factor-ß (TGF-ß) responses in C2C12 cells, suggesting increased sensitivity to TGF-ß in ICUAW muscle. Consistent with this suggestion, nuclear phospho-small mothers against decapentaplegic (SMAD) 2/3 was increased in ICUAW muscle. CONCLUSIONS: GDF-15 may increase sensitivity to TGF-ß signalling by suppressing the expression of muscle microRNAs, thereby promoting muscle atrophy in ICUAW. This study identifies both GDF-15 and associated microRNA as potential therapeutic targets.

Relationship between European eel Anguilla anguilla infection with non-native parasites and swimming behaviour on encountering accelerating flow.

The effect of Anguillicola crassus, Pseudodactylogyrus bini and Pseudodactylogyrus anguillae infection on the behaviour of downstream migrating adult European eels Anguilla anguilla as they encountered accelerating water velocity, common at engineered structures where flow is constricted (e.g. weirs and bypass systems), was evaluated in an experimental flume. The probability of reacting to, and rejecting, the velocity gradient was positively related to A. crassus larval, adult and total abundance. High abundance of Pseudodactylogyrus spp. reduced this effect, but A. crassus was the strongest parasitic factor associated with fish behaviour, and abundance was positively related to delay in downstream passage. Delayed downstream migration at hydraulic gradients associated with riverine anthropogenic structures could result in additional energetic expenditure for migrating A. anguilla already challenged by A. crassus infection.

Fibroblast cell therapy enhances initial healing in recessive dystrophic epidermolysis bullosa wounds: results of a randomized, vehicle-controlled trial.

BACKGROUND: Fibroblast cell therapy can modify disease biology in recessive dystrophic epidermolysis bullosa (RDEB) although whether it improves wound healing is not clear. OBJECTIVE: To assess the effects of injecting of allogeneic fibroblasts into the margins of chronic erosions in individuals with RDEB in a prospective, double-blind, randomized, vehicle-controlled phase II trial. METHODS: Erosions were randomized 1:1, to either a single treatment of 5 × 10(6) fibroblasts per linear cm of erosion margin or vehicle. All subjects continued standard wound care. The trial sponsor, participants and study outcome assessor were masked to treatment allocation. A hierarchy of endpoints germane to erosion closure was assessed. RESULTS: Twenty-six erosions in 11 subjects with RDEB were injected; 14 erosions received fibroblasts and 12 vehicle alone. A single series of injections was given at day 0 and all follow-up visits were completed. Treatment difference between fibroblasts and vehicle was -23.5% [95% confidence interval (CI) -3.5 to -43.5, P = 0.025] at day 7, -19.15% (95% CI 3.36 to -41.66, P = 0.089) at day 14 and -28.83% (95% CI 7.97 to -65.63, P = 0.11) at day 28. Beyond day 28, however, changes in mean erosion area did not differ significantly between the two groups. CONCLUSION: A single intradermal injection of allogeneic fibroblasts increases the initial rate of erosion healing in subjects with RDEB within the first 28 days but not thereafter. Further studies are needed to address the potential benefits of retreatment as well as optimal cell delivery.

Applying appropriate frequency criteria to advance acoustic behavioural guidance systems for fish.

Deterrents that use acoustics to guide fish away from dangerous areas depend on the elicitation of avoidance in the target species. Acoustic deterrents select the optimum frequency based on an assumption that highest avoidance is likely to occur at the greatest sensitivity. However, such an assumption may be unfounded. Using goldfish (Carassius auratus) as a suitable experimental model, this study tested this as a null hypothesis. Under laboratory conditions, the deterrence thresholds of individual goldfish exposed to 120 ms tones at six frequencies (250-2000 Hz) and four Sound Pressure Levels (SPL 115-145 dB) were quantified. The deterrence threshold defined as the SPL at which 25% of the tested population startled was calculated and compared to the hearing threshold obtained using Auditory Evoked Potential and particle acceleration threshold data. The optimum frequency to elicit a startle response was 250 Hz; different from the published hearing and particle acceleration sensitivities based on audiograms. The difference between the deterrence threshold and published hearing threshold data varied from 47.1 dB at 250 Hz to 76 dB at 600 Hz. This study demonstrates that information obtained from audiograms may poorly predict the most suitable frequencies at which avoidance behaviours are elicited in fish.

Boyle's Law ignores dynamic processes in governing barotrauma in fish.

The expansion and potential rupture of the swim bladder due to rapid decompression, a major cause of barotrauma injury in fish that pass through turbines and pumps, is generally assumed to be governed by Boyle's Law. In this study, two swim bladder expansion models are presented and tested in silico. One based on the quasi-static Boyle's Law, and a Modified Rayleigh Plesset Model (MRPM), which includes both inertial and pressure functions and was parametrised to be representative of a fish swim bladder. The two models were tested using a range of: (1) simulated and (2) empirically derived pressure profiles. Our results highlight a range of conditions where the Boyle's Law model (BLM) is inappropriate for predicting swim bladder size in response to pressure change and that these conditions occur in situ, indicating that this is an applied and not just theoretical issue. Specifically, these conditions include any one, or any combination, of the following factors: (1) when rate of pressure change is anything but very slow compared to the resonant frequency of the swim bladder; (2) when the nadir pressure is near or at absolute zero; and (3) when a fish experiences liquid tensions (i.e. negative absolute pressures). Under each of these conditions, the MRPM is more appropriate tool for predicting swim bladder size in response to pressure change and hence it is a better model for quantifying barotrauma in fish.

Pasture brome and perennial ryegrass characteristics that influence ewe lamb dietary preference during different seasons and periods of the day.

Under the current scenario for climate change, Bromus valdivianus Phil. (Bv), a drought-resistant species, is an option to complement Lolium perenne L. (Lp) in temperate pastures. However, little is known about animal preference for Bv. A randomised complete block design was used to study ewe lamb's preference between Lp and Bv during morning and afternoon grazing sessions in winter, spring, and summer by assessing the animal behaviour and pasture morphological and chemical attributes. Ewe lambs showed a higher preference for Lp in the afternoon in winter (P < 0.05) and summer (P < 0.01), while no differences were found in spring (P > 0.05). In winter, Bv, relative to Lp, had both greater ADF and NDF (P < 0.001), and lower pasture height (P < 0.01) which negatively affected its preference. The lack of differences in spring were due to an increase in ADF concentration in Lp. In summer, ewe lambs showed the typical daily preference pattern, selecting Lp in the morning to ensure a greater quality and showing no preference during the afternoon to fill the rumen with higher fibre content. In addition, greater sheath weight per tiller in Bv could make it less desirable, as the decrease in bite rate in the species was likely due to a higher shear strength and lower pasture sward mass per bite which increased foraging time. These results provided evidence on how Bv characteristics influence ewe lamb's preference; but more research is needed on how this will affect preference for Lp and Bv in a mixed pasture.

The effects of simulated hydropower turbine rapid decompression on two Neotropical fish species.

Barotrauma is a major cause of injury and mortality of fish as they pass through hydropower turbines. Current understanding of hydropower related barotrauma is biased towards northern temperate and southern subtropical species with single chambered swim bladders, specifically North American and Australian species, respectively. Today, unprecedented hydropower development is taking place in Neotropical regions where many species have complex multi-chambered swim bladder architecture. This study investigated barotrauma in two dual-chambered physostomous Neotropical fish (pacu, Piaractus mesopotamicus, and piracanjuba, Brycon orbignyanus) exposed to rapid (< 1 s) decompression at different Ratios of Pressure Change (RPC), using a hypo-hyperbaric chamber. The incidence and intensity (percentage surface area of organ affected) of injury and physiological and behavioural response (hereafter just response) of each species immediately after decompression was assessed. Twenty-two injury types (e.g. gill haemorrhage and exophthalmia) and eight response categories (e.g. rising to the surface and loss of orientation) were identified and the influence of: 1) species, 2) RPC, and 3) swim bladder rupture on each was quantified. There was considerable interspecific difference with emboli type injuries occurring more frequently in piracanjuba, but injury intensity tending to be higher in pacu. Both swim bladder chambers tended to rupture in piracanjuba but only the anterior chamber in pacu. RPC was positively correlated with response, incidence and intensity of several injury types for both species with some injuries occurring at very low RPC (e.g. 50 % probability of swim bladder rupture at 2.2 and 1.75 for piracanjuba and pacu, respectively). Multiple responses (e.g. loss of orientation) and injuries (e.g. eye haemorrhage) were correlated with swim bladder rupture suggesting gas venting into the body cavity likely causes secondary injury. When directly comparing our results with those available in the published literature, both pacu and piracanjuba appear to be more susceptible to barotrauma than previously studied subtropical and temperate species.

Molecular mechanisms of intensive care unit-acquired weakness.

Intensive care unit-acquired weakness (ICUAW) is an increasingly recognised and important clinical consequence of critical illness. It is associated with significant morbidity and mortality. The aetiology of this disease is not well understood. The purpose of this article is to review our understanding of the molecular pathogenesis of ICUAW in the context of current knowledge of clinical risk factors and aetiology. Key features of the disease are loss of muscle mass resulting from a shift in the dynamic balance of muscle protein synthesis and breakdown and a reduction in force-generating capacity. These alternations are secondary to neuropathy, disruption of the myofilament structure and function, a disrupted sarcoplasmic reticulum, electrical inexcitability and bioenergenetic failure. As knowledge and understanding of ICUAW grows, potential therapeutic targets will be identified, hopefully leading to multiple strategies for prevention and treatment of this important condition.

The serum concentration of active transforming growth factor-beta is severely depressed in advanced atherosclerosis.

Recent evidence has led us to propose that transforming growth factor-beta (TGF-beta) is a key inhibitor of atherosclerosis. We show here that a population of patients with advanced atherosclerosis all have less active TGF-beta in their sera than patients with normal coronary arteries, with a fivefold difference in average concentration between the two groups. This correlation with atherosclerosis is much stronger than for other known major risk factors and it may therefore have important diagnostic and prognostic significance. Aspirin medication correlates with an increase in active TGF-beta concentration, indicating that therapeutic interventions for TGF-beta are possible.

Experimental quantification of the swimming performance and behaviour of spawning run river lamprey Lampetra fluviatilis and European eel Anguilla anguilla.

Using a large-scale open-channel flume, the swimming ability and behaviour of individual adult European eel Anguilla anguilla and river lamprey Lampetra fluviatilis, species that exhibit anguilliform locomotion, were quantified under complex hydraulic conditions created by a 0·2-0·3 m high under- or overshot weir during four discharge regimes. Fishes were allowed to approach the weirs from both up- and downstream. All fishes passed the undershot weir, independent of discharge and direction of movement, and under high flow (mean ±S.E. 194·63 ± 6·48 l s(-1)) moved upstream against velocities that ranged between 1·75 and 2·12 m s(-1), suggesting greater maximum swimming capability than previously reported. In comparison, passage efficiency during upstream movement was lower for the overshot weir for both L. fluviatilis and A. anguilla. Downstream moving A. anguilla took longer to pass the over- than undershot weir. This study describes a methodology to attain realistic measures of swimming ability and behavioural performance required to develop multispecies fish passage criteria.

Role of Ucp1 enhancer methylation and chromatin remodelling in the control of Ucp1 expression in murine adipose tissue.

AIMS/HYPOTHESIS: Increasing the expression of the brown adipose tissue-specific gene uncoupling protein-1 (Ucp1) is a potential target for treating obesity. We investigated the role of DNA methylation and histone modification in Ucp1 expression in adipose cell lines and ex vivo murine adipose tissues. METHODS: Methylation state of the Ucp1 enhancer was studied using bisulphite mapping in murine adipose cell lines, and tissue taken from cold-stressed mice, coupled with functional assays of the effects of methylation and demethylation of the Ucp1 promoter on gene expression and nuclear protein binding. RESULTS: We show that demethylation of the Ucp1 promoter by 5-aza-deoxycytidine increases Ucp1 expression while methylation of Ucp1 promoter-reporter constructs decreases expression. Brown adipose tissue-specific Ucp1 expression is associated with decreased CpG dinucleotide methylation of the Ucp1 enhancer. The lowest CpG dinucleotide methylation state was found in two cyclic AMP response elements (CRE3, CRE2) in the Ucp1 promoter and methylation of the CpG in CRE2, but not CRE3 decreased nuclear protein binding. Chromatin immunoprecipitation assays revealed the presence of the silencing DiMethH3K9 modification on the Ucp1 enhancer in white adipose tissue and the appearance of the active TriMethH3K4 mark at the Ucp1 promoter in brown adipose tissue in response to a cold environment. CONCLUSIONS/INTERPRETATION: The results demonstrate that CpG dinucleotide methylation of the Ucp1 enhancer exhibits tissue-specific patterns in murine tissue and cell lines and suggest that adipose tissue-specific Ucp1 expression involves demethylation of CpG dinucleotides found in regulatory CREs in the Ucp1 enhancer, as well as modification of histone tails.

Strategic methodology to set priorities for sustainable hydropower development in a biodiversity hotspot.

Massive exploitation of freshwater systems for hydropower generation in developing countries is challenging sustainability due to cumulative environmental impacts in regions with high endemism. Habitat fragmentation is recognized as a major impact on river ecosystems. The nature and magnitude of connectivity loss depend on characteristics of the hydropower projects, and of the threatened fish communities. In areas where appropriate mitigation technology is lacking, there is a need to identify the fish species that are most at risk to better concentrate efforts. This paper aimed to set conservation priorities for sustainable hydropower development by analyzing native fish species and project characteristics. The Chilean ichthyogeographic province, an ecoregion with high endemism and massive hydropower projects development, has been considered as a case study. By using overlapping information on the characteristics of 1124 hydropower projects and distribution of native fish species, we identified three project categories of projects based on their need for mitigation. These were projects where mitigation was considered: a) not required (15%), b) required and feasible (35%), and c) required but challenging (50%). Projects where mitigation was not required were located at sites where native fish were absent and/or where water intakes allowed fish to pass. Interestingly, projects where mitigation was feasible were inhabited by a species assemblage that comprised the genus Trichomycterus, Diplomystes and Percilia, and the species Ch. pisciculus and B. maldonadoi. This finding emphasizes the need to develop a multispecific fishway that can accommodate this group. Projects where mitigation would be difficult to achieve were located at sites with a variety of different assemblages, thus making a standard fish pass solution challenging and site-specific. This study advances understanding for the need to develop mitigation strategies and technologies in ecoregions of high endemism threatened by hydropower and to prioritize the construction of planned projects.

Rethinking swimming performance tests for bottom-dwelling fish: the case of European glass eel (Anguilla anguilla).

Systematic experiments on European eel (Anguilla anguilla) in their juvenile, early life stage (glass eel), were conducted to provide new insights on the fish swimming performance and propose a framework of analysis to design swimming-performance experiments for bottom-dwelling fish. In particular, we coupled experimental and computational fluid dynamics techniques to: (i) accommodate glass eel burst-and-coast swimming mode and estimate the active swimming time (tac), not considering coast and drift periods, (ii) estimate near-bottom velocities (Ub) experienced by the fish, rather than using bulk averages (U), (iii) investigate water temperature (T) influence on swimming ability, and (iv) identify a functional relation between Ub, tac and T. Results showed that burst-and-coast swimming mode was increasingly adopted by glass eel, especially when U was higher than 0.3 ms-1. Using U rather than Ub led to an overestimation of the fish swimming performance from 18 to 32%, on average. Under the range of temperatures analyzed (from 8 to 18 °C), tac was strongly influenced and positively related to T. As a final result, we propose a general formula to link near-bottom velocity, water temperature and active swimming time which can be useful in ecological engineering applications and reads as [Formula: see text].

Effects of the polyamine spermine on arterial chemoreception.

Polyamines modulate many biological functions. Here we report a novel inhibitory modulation by spermine of catecholamine release by the rat carotid body and have identified the molecular mechanism underpinning it. We used molecular (RT-PCR and confocal microscopy) and functional (i.e., neurotransmitter release, patch clamp recording and calcium imaging) approaches to test the involvement of: (i) voltage-dependent calcium channels, and; (ii) the extracellular calcium-sensing receptor, CaR, a G protein-coupled receptor which is also activated by polyamines. RT-PCR and immunohistochemistry of isolated carotid bodies revealed that only Ca(v)1.2 and Ca(v)2.2 were expressed in type 1 cells while Ca(v)1.3, Ca(v)1.4, Ca(v)2.1, Ca(v)2.3 and Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3, could not be detected. CaR expression was detected exclusively in the nerve endings. In isolated carotid bodies, the hypoxia-dependent (7% O(2) for 10 minutes) and depolarization-evoked catecholamine release were partially suppressed by pre- (and co)-incubation with 500microM spermine. In dissociated type 1 glomus cells intracellular calcium concentration did not change following spermine treatment, but this polyamine did inhibit the depolarisation-evoked calcium influx. Whole-cell patch clamp recordings of HEK293 cells stably transfected with Ca(v)1.2 demonstrated that spermine inhibits this calcium channel. Interestingly, this inhibition was not apparent if the extracellular solution contained a concentration of Ba(2) above 2 mM as the charge carrier. In conclusion, spermine attenuates catecholamine release by the carotid body principally via inhibition of Ca(v)1.2. This mechanism may represent a negative feedback, which limits transmitter release during hypoxia.

Cysteine residues in the C-terminal tail of the human BK(Ca)alpha subunit are important for channel sensitivity to carbon monoxide.

In the presence of oxygen (O(2)), carbon monoxide (CO) is synthesised from heme by endogenous hemeoxygenases, and is a powerful activator of BK(Ca) channels. This transduction pathway has been proposed to contribute to cellular O(2) sensing in rat carotid body. In the present study we have explored the role that four cysteine residues (C820, C911, C995 and C1028), located in the vicinity of the "calcium bowl" of C-terminal of human BK(Ca)-alphasubunit, have on channel CO sensitivity. Mutant BK(Ca)-alphasubunits were generated by site-directed mutagenesis (single, double and triple cysteine residue substitutions with glycine residues) and were transiently transfected into HEK 293 cells before subsequent analysis in inside-out membrane patches. Potassium cyanide (KCN) completely abolished activation of wild type BK(Ca) channels by the CO donor, tricarbonyldichlororuthenium (II) dimer, at 100microM. In the absence of KCN the CO donor increased wild-type channel activity in a concentration-dependent manner, with an EC(50) of ca. 50microM. Single cysteine point mutations of residues C820, C995 and C1028 affected neither channel characteristics nor CO EC(50) values. In contrast, the CO sensitivity of the C911G mutation was significantly decreased (EC(50) ca. 100 M). Furthermore, all double and triple mutants which contained the C911G substitution exhibited reduced CO sensitivity, whilst those which did not contain this mutation displayed essentially unaltered CO EC(50) values. These data highlight that a single cysteine residue is crucial to the activation of BK(Ca) by CO. We suggest that CO may bind to this channel subunit in a manner similar to the transition metal-dependent co-ordination which is characteristic of several enzymes, such as CO dehydrogenase.