Building blue infrastructure: Assessing the key environmental issues and priority areas for ecological engineering initiatives in Australia's metropolitan embayments.

Ecological engineering principles are increasingly being applied to develop multifunctional artificial structures or rehabilitated habitats in coastal areas. Ecological engineering initiatives are primarily driven by marine scientists and coastal managers, but often the views of key user groups, which can strongly influence the success of projects, are not considered. We used an online survey and participatory mapping exercise to investigate differences in priority goals, sites and attitudes towards ecological engineering between marine scientists and coastal managers as compared to other stakeholders. The surveys were conducted across three Australian cities that varied in their level of urbanisation and environmental pressures. We tested the hypotheses that, relative to other stakeholders, marine scientists and coastal managers will: 1) be more supportive of ecological engineering; 2) be more likely to agree that enhancement of biodiversity and remediation of pollution are key priorities for ecological engineering; and 3) identify different priority areas and infrastructure or degraded habitats for ecological engineering. We also tested the hypothesis that 4) perceptions of ecological engineering would vary among locations, due to environmental and socio-economic differences. In all three harbours, marine scientists and coastal managers were more supportive of ecological engineering than other users. There was also greater support for ecological engineering in Sydney and Melbourne than Hobart. Most people identified transport infrastructure, in busy transport hubs (i.e. Circular Quay in Sydney, the Port in Melbourne and the Waterfront in Hobart) as priorities for ecological engineering, irrespective of their stakeholder group or location. There were, however, significant differences among locations in what people perceive as the key priorities for ecological engineering (i.e. biodiversity in Sydney and Melbourne vs. pollution in Hobart). Greater consideration of these location-specific differences is essential for effective management of artificial structures and rehabilitated habitats in urban embayments.

Making seawalls multifunctional: The positive effects of seeded bivalves and habitat structure on species diversity and filtration rates.

The marine environment is being increasingly modified by the construction of artificial structures, the impacts of which may be mitigated through eco-engineering. To date, eco-engineering has predominantly aimed to increase biodiversity, but enhancing other ecological functions is arguably of equal importance for artificial structures. Here, we manipulated complexity through habitat structure (flat, and 2.5 cm, 5 cm deep vertical and 5 cm deep horizontal crevices) and seeding with the native oyster (Saccostrea glomerata, unseeded and seeded) on concrete tiles (0.25 m × 0.25 m) affixed to seawalls to investigate whether complexity (both orientation and depth of crevices) influences particle removal rates by suspension feeders and colonisation by different functional groups, and whether there are any ecological trade-offs between these functions. After 12 months, complex seeded tiles generally supported a greater abundance of suspension feeding taxa and had higher particle removal rates than flat tiles or unseeded tiles. The richness and diversity of taxa also increased with complexity. The effect of seeding was, however, generally weaker on tiles with complex habitat structure. However, the orientation of habitat complexity and the depth of the crevices did not influence particle removal rates or colonising taxa. Colonisation by non-native taxa was low compared to total taxa richness. We did not detect negative ecological trade-offs between increased particle removal rates and diversity and abundance of key functional groups. Our results suggest that the addition of complexity to marine artificial structures could potentially be used to enhance both biodiversity and particle removal rates. Consequently, complexity should be incorporated into future eco-engineering projects to provide a range of ecological functions in urbanised estuaries.

Urban blue: A global analysis of the factors shaping people's perceptions of the marine environment and ecological engineering in harbours.

Marine harbours are the focus of a diverse range of activities and subject to multiple anthropogenically induced pressures. Support for environmental management options aimed at improving degraded harbours depends on understanding the factors which influence people's perceptions of harbour environments. We used an online survey, across 12 harbours, to assess sources of variation people's perceptions of harbour health and ecological engineering. We tested the hypotheses: 1) people living near impacted harbours would consider their environment to be more unhealthy and degraded, be more concerned about the environment and supportive of and willing to pay for ecological engineering relative to those living by less impacted harbours, and 2) people with greater connectedness to the harbour would be more concerned about and have greater perceived knowledge of the environment, and be more supportive of, knowledgeable about and willing to pay for ecological engineering, than those with less connectedness. Across twelve locations, the levels of degradation and modification by artificial structures were lower and the concern and knowledge about the environment and ecological engineering were greater in the six Australasian and American than the six European and Asian harbours surveyed. We found that people's perception of harbours as healthy or degraded, but not their concern for the environment, reflected the degree to which harbours were impacted. There was a positive relationship between the percentage of shoreline modified and the extent of support for and people's willingness to pay indirect costs for ecological engineering. At the individual level, measures of connectedness to the harbour environment were good predictors of concern for and perceived knowledge about the environment but not support for and perceived knowledge about ecological engineering. To make informed decisions, it is important that people are empowered with sufficient knowledge of the environmental issues facing their harbour and ecological engineering options.

Characterization of delta opioid receptors in lung cancer using a novel nonpeptidic ligand.

Cancer cells are often characterized by the presence of membrane receptors not normally associated with nontransformed cells from the same tissue type. Recent studies have demonstrated increased expression of high-affinity binding sites for opioid receptor-selective ligands in lung cancer cell lines relative to normal lung tissue. We investigated the binding of a nonpeptidic delta opioid receptor ligand in small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells with the aim of developing the ligand as a novel lung cancer imaging agent. The ligand, [3H] (+)-4-[alpha-R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3- hydroxybenzyl)-N,N-diethylbenzamide ([3H](+)BW373U86), bound with high-affinity [Kd (dissociation constant) = 0.066 +/- 0.012 nM] to membranes prepared from six different SCLC cell lines but not to those from seven NSCLC cell lines, including one mesothelioma. The number of biding sites varied from 10 to 300 fmol/mg membrane protein. Competition binding studies demonstrated displacement of [3H](+)BW373U86 binding by the delta-selective antagonists naltriben and 7-benzylidenenaltrexone but not with the mu- and kappa- selective antagonists D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 and trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]ben zeneacetamide methanesulfonate. Mean apparent Kis for naltriben and 7-benzylidenenaltrexone in membranes from two SCLC cell lines were 0.17 and 3.9 nM, respectively, but were >10 microM for the mu and kappa ligands. The nonselective antagonist naloxone displaced [3H](+)BW373U86 binding with an apparent Ki of approximately 29 nM. On the basis of these data, we believe the lung cancer receptor to be similar, if not identical, to the human brain delta opioid receptor. The lack of high-affinity [3H](+)BW373U86 binding in normal mouse lung membranes suggests a potential role for this ligand as a novel therapeutic or imaging agent.

Image-Based Personalization of Cardiac Anatomy for Coupled Electromechanical Modeling.

Computational models of cardiac electromechanics (EM) are increasingly being applied to clinical problems, with patient-specific models being generated from high fidelity imaging and used to simulate patient physiology, pathophysiology and response to treatment. Current structured meshes are limited in their ability to fully represent the detailed anatomical data available from clinical images and capture complex and varied anatomy with limited geometric accuracy. In this paper, we review the state of the art in image-based personalization of cardiac anatomy for biophysically detailed, strongly coupled EM modeling, and present our own tools for the automatic building of anatomically and structurally accurate patient-specific models. Our method relies on using high resolution unstructured meshes for discretizing both physics, electrophysiology and mechanics, in combination with efficient, strongly scalable solvers necessary to deal with the computational load imposed by the large number of degrees of freedom of these meshes. These tools permit automated anatomical model generation and strongly coupled EM simulations at an unprecedented level of anatomical and biophysical detail.

Maximum likelihood alignment of DNA sequences.

The optimal alignment problem for pairs of molecular sequences under a probabilistic model of evolutionary change is equivalent to the problem of estimating the maximum likelihood time required to transform one sequence to the other. When this time has been estimated, various alignments of high posterior probability may be written down. A simple model with two parameters is presented and a method is described by which the likelihood may be computed. Maximum likelihood estimates for some pairs of tRNA genes illustrate the method and allow us to obtain the best alignments under the model.

The identification and characterization of microsatellites in the compact genome of the Japanese pufferfish, Fugu rubripes: perspectives in functional and comparative genomic analyses.

Fugu rubripes (Fugu) has one of the smallest recorded vertebrate genomes and is an economic tool for comparative DNA sequence analysis. Initial characterization of 128 kb of Fugu DNA attributed the compactness of this genome, in part, to a sparseness of repetitive DNA sequence compared with mammalian genomic sequences. This paper describes a new and comprehensive analysis in which 501 theoretically possible microsatellites with a repeat unit of one to six bases were used to query two orders of magnitude more Fugu DNA (i.e. 11.338 Mb). A total of 6042 microsatellites were identified and categorized. In decreasing order, the 20 most frequently occurring microsatellites are AC, A, C, AGG, AG, AGC, AAT, AAAT, ACAG, ACGC, ATCC, AAC, ATC, AGGG, AAAG, AAG, AAAC, AT, CCG and TTAGGG. The 20 most frequently occurring microsatellites represent 81.79% of all microsatellites identified. Our results indicate that one microsatellite occurs every 1.876 kb of DNA in Fugu, 11.55% of the microsatellites are detected in open reading frames that are predicted protein coding regions. With respect to the proportion of microsatellites present in open reading frames and the total abundance (bp) of all microsatellites, the genome of Fugu is similar to the genome of many other vertebrate species. Previous estimates performed indicate that approximately 1% of many vertebrate genomes are comprized of microsatellite sequences. However, many differences prevail in the abundance and frequency of the individual microsatellite classes. Many of the frequently occurring microsatellites in Fugu are known to code in other species for regions in proteins such as transcription factors, whilst others are associated with known functions, such as transcription factor binding sites and form part of promoter regions in DNA sequences of genes. Therefore, it is likely that such repeats in genomes have a role in the evolution of genes, regulation of gene expression and consequently the evolution of species.

Pressures required to move gas through the native airway in the presence of a fenestrated vs a nonfenestrated tracheostomy tube.

SUBJECT: It is occasionally desirable for patients with a tracheostomy tube to breathe through their native airway. We hypothesized that capped tracheostomy tubes with cuffs deflated would create substantial additional resistance to airflow without fenestration but would provide minimal resistance to airflow when the tube had a fenestration. METHODS: Two tracheal models were tested simulating a large (26 mm) and an average (18 mm) trachea. Tests were carried out with fenestrated and nonfenestrated tracheostomy tubes of sizes ranging from No. 4 to No. 10. Negative pressure inspiration was simulated using suction. RESULTS: With a large trachea or small tubes, the suction required to generate flows of 40 L/min or greater remained less than 5 cm H2O with or without a fenestration. However, with an average-sized trachea and no fenestration, the pressure required to generate flows of 40 L/min or greater exceeded 5 cm H2O and with No. 8 or No. 10 tubes exceeded 20 cm H2O. A fenestration routinely reduced the required pressure to less than 5 cm H2O. CONCLUSION: The effort required to move gas across the native airway in the absence of a fenestration may be substantial. If a patient is to breathe through a native airway, a fenestrated tube should be used unless the tracheostomy tube is a No. 4.

Head position and oral vs nasal route as factors determining endotracheal tube resistance.

STUDY OBJECTIVE: We performed this in vitro study to determine the following: (1) if there is any significant difference in resistance between comparably sized endotracheal tubes (ETTs) in simulated anatomic oral and nasal conformations: (2) if neck flexion would increase the resistance of the ETT; and (3) if a wire-reinforced tube in simulated oral conformation would minimize the resistance increase at bends in the tube. DESIGN: The pressure drops (the change in pressure the flow through the ETT) at the proximal end of three sizes of tubes (6-, 7-, and 8-mm inner diameter) were measured in anatomic conformations at flows ranging from 20 to 100 L/min with the tubes warmed to 37 degrees C. RESULTS: There were no significant differences in pressure drops between comparably sized ETTs in the nasal vs oral conformation at any flow tested. Maximal head flexion produced a small increase in pressure drop for the standard 6-mm ETT but not for the larger tubes. The wire-reinforced tubes, when compared with the standard 7- and 8-mm ETTS, actually had greater pressure drops across the tubes at high flows. CONCLUSIONS: (1) Pressure differences between ETTs in nasal and oral conformations with comparable size and length are insignificant, even at high flow rates. (2) Maximal neck flexion does not deform the tube enough to make a clinically significant difference in resistance in vitro, although the 6-mm ETT had a slightly higher resistance in flexion. (3) A size 7- or 8-mm reinforced ETT has a higher resistance compared with a standard ETT.

Carbon dioxide excretion via bronchopleural fistulas in adult respiratory distress syndrome.

Expired gas and gas leaked via thoracostomy tubes was collected in nine patients with the adult respiratory distress syndrome (ARDS) and bronchopleural fistulas. Mean minute ventilation in the group was 23.9 +/- 7.5 L/min with 31 +/- 23 percent of the gas escaping via the leak. Carbon dioxide was present in the leaked gas in every patient, with the fraction of CO2 excreted via the leak highly correlated with the fraction of minute ventilation exiting via the leak (r = 0.86, p less than 0.005). Mean concentration of CO2 in leaked gas was 1.3 +/- 0.5 percent. We conclude that some of the gas leaked via a bronchopleural fistula in such patients participated in gas exchange.

Dexamethasone treatment does not ameliorate subglottic ischemic injury in rabbits.

BACKGROUND: Following tracheal intubation, a small proportion of patients develop laryngeal inflammation or tissue necrosis severe enough to result in clinical symptoms. Although corticosteroids are frequently advocated to prevent such injury, human studies have been inconclusive because of the low incidence of the problem. This study developed a rabbit model of endotracheal tube-induced laryngeal injury to test the hypothesis that a corticosteroid, dexamethasone, could ameliorate the inflammation and necrosis. METHODS: Subglottic injury was induced in 21 anesthetized rabbits by inflating the cuff of an endotracheal tube to 100 mm Hg with the cuff just below the vocal cords. Every 30 min for 2 h, the cuff was deflated, the tube turned 90 degrees, and the cuff then reinflated. After 2 h, the rabbits' tracheas were extubated. Rabbits were divided into two groups: the treatment group received dexamethasone (1 mg/kg) i.v. 1 h prior to extubation with the dose repeated 6 h following extubation; the untreated group received a saline solution placebo. Four additional rabbits were anesthetized for the same period but did not have a tracheal tube inserted. All rabbits were killed 24 h later and the larynxes were harvested. Sections through the larynx at the level of the cricoid cartilage were randomized and submitted blindly to a veterinary pathologist. Larynxes were scored and ranked according to the severity of mucosal inflammation and necrosis, and submucosal hemorrhage, edema, inflammation, and necrosis. Specimens were also evaluated for focal vs diffuse disease. RESULTS: Injured rabbits demonstrated focal to diffuse mucosal and submucosal inflammation and necrosis. Inflammatory exudates were present in sections from most of the injured rabbits and large sections of the larynxes were denuded of epithelium. There were no differences in injury scores between the treated and untreated rabbits. The four uninjured control rabbits had normal larynxes. CONCLUSIONS: Two hours of endotracheal tube cuff inflation to 100 mm Hg causes an inflammatory laryngeal injury. The histologic features of the injury are unaltered by treatment with 2 mg/kg dexamethasone.

Effect of anemia on intrapulmonary shunt during atelectasis in rabbits.

To elucidate the effects of anemia on intrapulmonary shunt, we studied a model of left lung atelectasis in anesthetized rabbits. In 10 rabbits, isovolemic anemia was produced by sequential hemodilution. Seven control rabbits were followed over time, without hemodilution. Intrapulmonary shunt (Qs/QT) was measured by using blood gas analysis and by quantitation of the percentage of blood flow to the collapsed left lung (QLl/QT) using fluorescent microspheres. In control rabbits, Qs/QT and QLl/QT decreased over time, whereas arterial PO2 increased. In hemodiluted rabbits, there was a trend toward increased Qs/QT and QLl/QT. There were significant differences in the behavior of Qs/QT, QLl/QT, and arterial PO2 between control and hemodiluted rabbits. Hemodynamic parameters, including cardiac output and pulmonary artery pressure, were not different between groups. In a third group of rabbits with pharmacologically induced acidosis but no hemodilution, Qs/QT and QLl/QT decreased over time, and arterial PO2 increased. We conclude that acute isovolemic anemia has a deleterious effect on pulmonary gas exchange, possibly through attenuation of hypoxic pulmonary vasoconstriction.

Lung reperfusion in dogs causes bilateral lung injury.

Occlusion of the pulmonary arterial circulation to a lung for prolonged periods has been reported to result in only minimal alterations in lung morphology. We studied the effects of 48 h of pulmonary arterial occlusion followed by 4 h of reperfusion in 18 awake dogs. Because of evidence in other organ systems of O2 radical generation, during reperfusion, nine of the animals were randomly assigned to receive allopurinol, a xanthine oxidase inhibitor, and vitamin E, an antioxidant. Reperfusion resulted in marked edema and inflammatory infiltrates in the reperfused lung but also caused mild edema and inflammation in the contralateral continuously perfused lung. Electron microscopy demonstrated lysis of both capillary endothelial and alveolar epithelial cells bilaterally, with the frequency of cell injury greater on the reperfused side. During reperfusion, body temperatures rose dramatically from 39.4 +/- 0.1 to 40.6 +/- 0.2 degrees C (P less than 0.05) and marked leukopenia developed. There were no differences in any hemodynamic, gas exchange, or morphometric measurements between allopurinol-treated dogs and untreated animals. We conclude that reperfusion causes local and distant injury which does not appear to be mediated by xanthine oxidase-produced O2 radicals.

Pulmonary artery occlusion is sufficient to increase pulmonary vascular permeability in rabbits.

Unilateral pulmonary artery obstruction (PAO) for 24-48 h, followed by reperfusion, results in pulmonary edema and lung inflammation. We hypothesized that lung injury actually occurred during the period of PAO but, because of low microvascular pressures during the period of occlusion, was not detected until perfusion was reestablished. To test this hypothesis, we studied 14 rabbits divided into three groups: group I rabbits underwent sham occlusion of the left pulmonary artery for 24 h; group II rabbits underwent PAO but were not reperfused; and group III rabbits were subjected to PAO and then reperfused for 4 h. The fluid filtration coefficient measured during a zone 3 no-flow hydrostatic stress (pulmonary arterial pressure = pulmonary venous pressure, both greater than alveolar pressure) in group I lungs was less than that of lungs in either group II or III [0.52 +/- 0.02 (SE) ml.min-1.cmH2O.100 g wet wt-1 vs. 0.94 +/- 0.11 and 0.86 +/- 0.13 for groups II and III, respectively, P less than 0.05]. The wet-to-dry weight ratio of the left lung measured after the zone 3 stress was applied for 20 min was 6.90 +/- 0.09 in group I rabbits and 9.21 +/- 0.75 and 11.75 +/- 0.44 in groups II and III, respectively (P less than 0.05). Radiolabeled microspheres demonstrated that flow to the left lung was diminished after the period of PAO (38 +/- 4, 9 +/- 5, and 2 +/- 1% of cardiac output in groups I, II, and III, respectively; P less than 0.05 for group I vs. groups II and III).(ABSTRACT TRUNCATED AT 250 WORDS)

Dimethylthiourea does not ameliorate reperfusion lung injury in dogs or rabbits.

We previously demonstrated that in vivo reperfusion of a dog lung after 48 h of pulmonary arterial (PA) ischemia results in pulmonary edema with a significant infiltrate of polymorphonuclear leukocytes. We hypothesized that the injury resulted from production of hydroxyl radical by activated neutrophils. In the current study, we attempted to prevent the injury in both dogs and rabbits with dimethylthiourea (DMTU), a scavenger of hydroxyl radical. After 48 h of left PA occlusion in 18 dogs, DMTU was administered to 9 animals and 9 were not treated. The occlusion was then released, and the dogs were killed 4 h later. Reperfusion resulted in a drop in leukocyte count and left lung edema, but there was no difference between treated and untreated animals. The wet-to-dry ratios of the lungs in the treated group were 5.76 +/- 0.44 (SE) on the reperfused left side and 4.50 +/- 0.06 (P less than 0.05) on the right side. In the untreated groups the comparable ratios were 5.73 +/- 0.31 and 4.92 +/- 0.10 (P less than 0.05 for right vs. left). Histological examination revealed significant differences between the right and left lungs in the extent of intra-alveolar granulocytes and macrophages but did not reveal differences between the treated and untreated animals. To ensure that neither the model nor the lack of response to DMTU was species specific, we then developed a rabbit model of reperfusion edema.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional alveolar hypoxia does not affect air embolism-induced pulmonary edema.

We studied the effects of regional alveolar hypoxia on permeability pulmonary edema resulting from venous air embolization. Anesthetized dogs had the left upper lobe removed and a double-lumen tube placed so that right lung and left lower lobe (LLL) could be ventilated independently. Air was infused into the femoral vein for 1 h during bilateral ventilation at an inspiratory O2 fraction (FIO2) of 1.0. After cessation of air infusion the LLL was then ventilated with a hypoxic gas mixture (FIO2 = 0.05) in six animals and an FIO2 of 1.0 in six other animals. Lung hydroxyproline content was measured as an index of lung dry weight. LLL bloodless lobar wet weight-to-hydroxyproline ratio was 0.33 +/- 0.06 mg/micrograms in the animals exposed to LLL hypoxia and 0.37 +/- 0.03 mg/micrograms (NS) in the animals that had a LLL FIO2 of 1. Both values were significantly higher than our laboratory normal values of 0.19 +/- 0.01 mg/micrograms. We subsequently found in four more dogs exposed to global alveolar hypoxia before and after air embolism that the air injury itself significantly depressed the hypoxic vasoconstrictor response. We conclude that regional alveolar hypoxia has no effect on pulmonary edema formation due to air embolism. The most likely reason for these findings is that the air embolism injury itself interfered with hypoxic pulmonary vasoconstriction.

Hypoxic pulmonary vasoconstriction does not affect hydrostatic pulmonary edema formation.

We studied the effects of regional hypoxic pulmonary vasoconstriction (HPV) on lobar flow diversion in the presence of hydrostatic pulmonary edema. Ten anesthetized dogs with the left lower lobe (LLL) suspended in a net for continuous weighing were ventilated with a bronchial divider so the LLL could be ventilated with either 100% O2 or a hypoxic gas mixture (90% N2-5% CO2-5% O2). A balloon was inflated in the left atrium until hydrostatic pulmonary edema occurred, as evidenced by a continuous increase in LLL weight. Left lower lobe flow (QLLL) was measured by electromagnetic flow meter and cardiac output (QT) by thermal dilution. At a left atrial pressure of 30 +/- 5 mmHg, ventilation of the LLL with the hypoxic gas mixture caused QLLL/QT to decrease from 17 +/- 4 to 11 +/- 3% (P less than 0.05), pulmonary arterial pressure to increase from 35 +/- 5 to 37 +/- 6 mmHg (P less than 0.05), and no significant change in rate of LLL weight gain. Gravimetric confirmation of our results was provided by experiments in four animals where the LLL was ventilated with an hypoxic gas mixture for 2 h while the right lung was ventilated with 100% O2. In these animals there was no difference in bloodless lung water between the LLL and right lower lobe. We conclude that in the presence of left atrial pressures high enough to cause hydrostatic pulmonary edema, HPV causes significant flow diversion from an hypoxic lobe but the decrease in flow does not affect edema formation.

Effect of regional alveolar hypoxia on permeability pulmonary edema formation in dogs.

We studied the effects of regional alveolar hypoxia on permeability pulmonary edema formation. Anesthetized dogs had a bronchial divider placed so that the left lower lobe (LLL) could be ventilated with a hypoxic gas mixture (HGM) while the right lung was continuously ventilated with 100% O2. Bilateral permeability edema was induced with 0.05 ml/kg oleic acid and after 4 h of LLL ventilation with an HGM (n = 9) LLL gross weight was 161 +/- 13 (SE) g compared with 204 +/- 13 (SE) g (P less than 0.05) in the right lower lobe (RLL). Bloodless lobar water and dry weight were also significantly lower in the LLL as compared with the RLL of the study animals. In seven control animals in which the LLL fractional inspired concentration of O2 (FIO2) was 1.0 during permeability edema, there were no differences in gravimetric variables between LLL and RLL. In eight additional animals, pulmonary capillary pressure (Pc), measured by simultaneous occlusion of left pulmonary artery and vein, was not significantly different between LLL FIO2 of 1.0 and 0.05 either before or after pulmonary edema. We conclude that, in the presence of permeability pulmonary edema, regional alveolar hypoxia causes reduction in edema formation. The decreased edema formation during alveolar hypoxia is not due to a reduction in Pc.