Rapid, economical single-nucleotide polymorphism and microsatellite discovery based on de novo assembly of a reduced representation genome in a non-model organism: a case study of Atlantic cod Gadus morhua.

By combining next-generation sequencing technology (454) and reduced representation library (RRL) construction, the rapid and economical isolation of over 25 000 potential single-nucleotide polymorphisms (SNP) and >6000 putative microsatellite loci from c. 2% of the genome of the non-model teleost, Atlantic cod Gadus morhua from the Celtic Sea, south of Ireland, was demonstrated. A small-scale validation of markers indicated that 80% (11 of 14) of SNP loci and 40% (6 of 15) of the microsatellite loci could be amplified and showed variability. The results clearly show that small-scale next-generation sequencing of RRL genomes is an economical and rapid approach for simultaneous SNP and microsatellite discovery that is applicable to any species. The low cost and relatively small investment in time allows for positive exploitation of ascertainment bias to design markers applicable to specific populations and study questions.

Embryonic development in ballan wrasse Labrus bergylta.

Eight primary embryonic developmental stages were assigned to eggs of ballan wrasse Labrus bergylta using key morphological features following standardized nomenclature: Ia, Ib, II, III, IV, V, VI and VI+, reared from single family clutches under comparable environmental conditions in Ireland and Norway. Development in L. bergylta is typical of demersal marine finfish species with a short egg stage. Hatching occurred c. 123 h post-fertilization (hpf) equivalent to 62·5 degree days at 12·2 ± 1·10° C (mean ±S.D.), after which the larvae swam intermittently near the surface of the water column.

Drivers of ecological assembly in the hindgut of Atlantic Cod fed a macroalgal supplemented diet.

It is difficult to disentangle the many variables (e.g. internal or external cues and random events) that shape the microbiota in the gastrointestinal tract of any living species. Ecological assembly processes applied to microbial communities can elucidate these drivers. In our study, farmed Atlantic cod (Gadus morhua) were fed a diet of 10% macroalgae supplement (Ulva rigida [ULVA] or Ascophyllum nodosum [ASCO] or a non-supplemented control diet [CTRL]) over 12 weeks. We determined the influence of ecological assembly processes using a suite of null-modelling tools. We observed dissimilarity in the abundance of common OTUs over time, which was driven by deterministic assembly. The CTRL samples showed selection as a critical assembly process. While dispersal limitation was a driver of the gut microbiome for fish fed the macroalgae supplemented diet at Week 12 (i.e., ASCO and ULVA). Fish from the ASCO grouping diverged into ASCO_N (normal) and ASCO_LG (lower growth), where ASCO_LG individuals found the diet unpalatable. The recruitment of new taxa overtime was altered in the ASCO_LG fish, with the gut microbiome showing phylogenetic underdispersion (nepotistic species recruitment). Finally, the gut microbiome (CTRL and ULVA) showed increasing robustness to taxonomic disturbance over time and lower functional redundancy. This study advances our understanding of the ecological assembly and succession in the hindgut of juvenile Atlantic cod across dietary treatments. Understanding the processes driving ecological assembly in the gut microbiome, in fish research specifically, could allow us to manipulate the microbiome for improved health or resilience to disease for improved aquaculture welfare and production.

Temporal changes in the gut microbiota in farmed Atlantic cod (Gadus morhua) outweigh the response to diet supplementation with macroalgae.

BACKGROUND: Aquaculture successfully meets global food demands for many fish species. However, aquaculture production of Atlantic cod (Gadus morhua) is just 2.5% of total market production. For cod farming to be a viable economic venture specific challenges on how to increase growth, health and farming productivity need to be addressed. Feed ingredients play a key role here. Macroalgae (seaweeds) have been suggested as a functional feed supplement with both health and economic benefits for terrestrial farmed animals and fish. The impact of such dietary supplements to cod gut integrity and microbiota, which contribute to overall fish robustness is unknown. The objective of this study was to supplement the diet of juvenile Atlantic cod with macroalgae and determine the impacts on fish condition and growth, gut morphology and hindgut microbiota composition (16S rRNA amplicon sequencing). Fish were fed one of three diets: control (no macroalgal inclusion), 10% inclusion of either egg wrack (Ascophyllum nodosum) or sea lettuce (Ulva rigida) macroalgae in a 12-week trial. RESULTS: The results demonstrated there was no significant difference in fish condition, gut morphology or hindgut microbiota between the U. rigida supplemented fish group and the control group at any time-point. This trend was not observed with the A. nodosum treatment. Fish within this group were further categorised as either 'Normal' or 'Lower Growth'. 'Lower Growth' individuals found the diet unpalatable resulting in reduced weight and condition factor combined with an altered gut morphology and microbiome relative to the other treatments. Excluding this group, our results show that the hindgut microbiota was largely driven by temporal pressures with the microbial communities becoming more similar over time irrespective of dietary treatment. The core microbiome at the final time-point consisted of the orders Vibrionales (Vibrio and Photobacterium), Bacteroidales (Bacteroidetes and Macellibacteroides) and Clostridiales (Lachnoclostridium). CONCLUSIONS: Our study indicates that U. rigida macroalgae can be supplemented at 10% inclusion levels in the diet of juvenile farmed Atlantic cod without any impact on fish condition or hindgut microbial community structure. We also conclude that 10% dietary inclusion of A. nodosum is not a suitable feed supplement in a farmed cod diet.

Effectiveness of nitric oxide inhalation in septic ARDS.

STUDY OBJECTIVE: To evaluate the percentage of nitric oxide (NO) responders in septic shock patients with ARDS. Additionally, to investigate long-term NO effects on cardiac performance and oxygen kinetic patterns in NO responders vs nonresponders. DESIGN: Prospective cohort study. SETTING: ICU of a university hospital. PATIENTS: Twenty-five consecutive patients with a diagnosis of septic shock and established ARDS requiring inotropic and vasopressor support. INTERVENTIONS: After diagnosis of ARDS, NO was administered at 18 or 36 ppm. Patients demonstrating a NO-induced rise of arterial oxygen tension of 20% or more and/or a fall in mean pulmonary artery pressure of 15% or more were grouped as NO responders; others were grouped as nonresponders. MEASUREMENTS AND RESULTS: Ten patients (40%) were NO responders, while 15 patients (60%) were nonresponders. Mortality was 40% in NO responders and 67% in nonresponders (NS). NO responders developed a significantly lower mean pulmonary artery pressure (28 +/- 6 vs 33 +/- 6 mm Hg; p < 0.05), lower pulmonary vascular resistance (PVR: 258 +/- 73 vs 377 +/- 163 dyne.s.cm-5.m-2; p < 0.05), and higher PaO2/FIO2 ratio (192 +/- 85 vs 144 +/- 74 mm Hg; p < 0.05) within the study period. In responders, NO-induced afterload reduction resulted in increased right ventricular ejection fraction (RVEF: 40 +/- 7 vs 35 +/- 9%; p < 0.05), significantly higher cardiac index (CI: 4.5 +/- 1.1 vs 4.0 +/- 1.2 L.min-1.m-2; p < 0.05) and oxygen delivery (DO2: 681 +/- 141 vs 599 +/- 160 mL.min-1.m-2; p < 0.05) compared with nonresponders. In NO nonresponders, RVEF was correlated with PVR, CI, DO2, mixed venous oxygen saturation (SvO2), and oxygen extraction ratio (O2ER) (r = +/- 0.60 to +/- 0.69; p < 0.05). No significant correlation between RVEF and any of these parameters was observed in responders. SvO2 (75 +/- 7 vs 69 +/- 8%; p < 0.05) and O2ER (0.24 +/- 0.06 vs 0.27 +/- 0.06; p < 0.05) were significantly different between responders and nonresponders, while no difference in oxygen consumption was observed (161 +/- 41 vs 153 +/- 43 mL.min.m-2). CONCLUSIONS: Inhaled NO is effective in only a subgroup of septic ARDS patients, with a higher, but insignificantly different percentage of survivors in the responder group. NO responders were characterized by increased RVEF accompanied by higher CI, DO2, and lower O2ER. In nonresponders, RVEF remained depressed, with a close correlation between RVEF and CO as well as DO2 and O2ER. Thus, nonresponders seem to suffer from impaired cardiac reserves and correspondingly lower oxygen transport variables.

Morphine influences on classical aversive conditioned heart rate in rats.

The present series of three experiments was concerned with the effects of morphine and the morphine antagonist naloxone on the development of a classical aversive heart rate (HR) conditioned response (CR) to a tone conditioned stimulus (CS) paired with an electric shock unconditioned stimulus (US). In the first study, separate groups of rats received preconditioning sc injections of either 0.25 mg/kg, 5 mg/kg, or 10 mg/kg of morphine. Three other groups were given 0.1 mg/kg, 5 mg/kg, or 10 mg/kg of naloxone alone. All of the morphine groups showed attenuation HR responses to the CS on preconditioning CS-alone trials. During conditioning, the 10-mg/kg morphine group showed a markedly decremented bradycardia CR and tachycardia unconditioned response (UR), whereas the 5-mg/kg morphine group showed a normal CR in combination with a decremented UR. Naloxone had no measurable effects on HR. In the second study, naloxone (1 mg/kg) given after conditioning failed to reverse the CR and UR losses produced by 10 mg/kg of morphine given prior to conditioning. Administration of 10 mg/kg of morphine produced only a minor reduction in a HR CR established in a drug-free state, but the tachycardia UR was severely reduced. The results of the third study showed that 1 mg/kg of naloxone was effective in reversing analgesia induced by 10 mg/kg of morphine, as indexed by the tail-flick test. Taken together, the results suggest that the 10-mg/kg dose of morphine interfered with the learning of a HR CR, perhaps principally by reducing the aversive or emotional consequences of the shock US. Direct cardiovascular effects of morphine seemed to interfere with the performance of the tachycardia UR, but not with the performance of the bradycardia CR.

Effects of centrally administered anxiolytic agents on classically conditioned bradycardia.

Rats received infusions of the opioid peptide D-Ala2-Met-enkephalinamide (DALA, 10 micrograms), the alpha 2-noradrenergic agonist clonidine (CLON, 3 micrograms), UK14,304 (UK, 5 micrograms), the corticotropin-releasing factor (CRF) antagonist alpha-helical CRF (9-41) (alpha-HEL, 25 micrograms), or saline in the rostral fourth ventricle. The DALA, CLON, and UK groups showed no evidence of a heart rate (HR) conditioned response (CR) during conditioning, after antagonist administration, or on a nondrug test 48 hr after conditioning. These three groups showed the development of normal CRs when later retrained without drugs. The alpha-HEL group showed an enhanced CR. During a subsequent startle test, the presence of a conditioned stimulus resulted in a pronounced suppression of startle in the SAL and alpha-HEL groups but had no effects on startle in the DALA, CLON, and UK groups. The results indicate an important role for fourth ventricle structures containing opioid and alpha 2 receptors in the learning of an HR CR.

Impaired learning of classically conditioned bradycardia in rats following fourth ventricle administration of D-Ala2-methionine-enkephalinamide.

Prior to differential classical conditioning on two successive days, three groups of rats received an infusion (10 micrograms) of either the opioid peptide D-alanine2-methionine-enkephalinamide (DALA), DALA plus naltrexone (5 micrograms), or saline into the rostral region of the fourth ventricle. A fourth group, which served as a control to help localize DALA's site of action, received an infusion of DALA (10 micrograms) into the brain stem area on the floor of the ventricle. The group given DALA alone in the ventricle showed no evidence of a heart rate conditioned response (CR) either during conditioning or during a nondrug test session given 2 days after conditioning. Interference with the CR by DALA was reversed by the concomitant infusion of naltrexone. The control group given DALA in the brain stem developed a normal CR. It was suggested that DALA-induced opioid-receptor activity in the region of the periaqueductal/periventricular gray or locus coeruleus region of the ventricle may have prevented the learning of a CR. This could have occurred through a blunting of the emotional aftereffects of the unconditioned stimulus or through interference with projection pathways to other areas.

Effects of drug-induced changes in resting blood pressure on classically conditioned heart rate and blood pressure in restrained rats.

Subsequent to receiving aversive classical conditioning, which led to a decelerative heart rate (HR) conditioned response (CR) and a pressor-depressor blood pressure (BP) CR, three separate groups of restrained rats received intravenous infusion of sodium nitroprusside (40 micrograms/mg/min) to lower baseline BP, phenylephrine (17 micrograms/mg/min) to raise baseline BP, or an equivalent volume of saline. Conditioning test trials during infusion revealed that hypotension produced by sodium nitroprusside eliminated the HR CR and transformed the BP CR into a pressor-only reaction. Hypertension produced by phenylephrine facilitated the HR CR and changed the BP CR to a pressor-only response on selected trials in which baseline BP increases and baseline HR decreases were within restricted limits. Following drug withdrawal, the HR CRs of both drug groups and the BP CR of the phenylephrine group were attenuated. The unconditioned responses to the shock unconditioned stimulus under phenylephrine were exaggerated and consisted of tachycardias and depressor BP changes, whereas under sodium nitroprusside reduced tachycardias and depressor activity occurred. The results suggested that the loss of the vagally mediated HR CR under sodium nitroprusside was due to baroreceptor-controlled inhibition of vagal discharge and that the enhancement of the HR CR under phenylephrine was due to baroreceptor-influenced facilitation of vagal discharge.

Learning versus performance effects of cocaine on discriminative heart rate conditioning in rats.

The study examined the effects of cocaine on learning and performance of a classically conditioned heart rate (HR) discrimination in rats involving two auditory conditioned stimuli (CSs). In the discrimination protocol, one CS (CS+) was paired with the shock unconditioned stimulus (US) on a consistent basis and the other CS (CS-) was always presented alone. Four groups received an IP injection of 1, 3, 10, or 30 mg/kg cocaine and a fifth group received saline. Shortly after the injections, all groups were given six CS-alone trials, followed by 24 randomly sequenced discrimination conditioning trials (12 CS+ and 12 CS-). Approximately 72 h later, all groups were given six test trials with each CS in the absence of cocaine to evaluate the presence or absence of discrimination learning. All cocaine groups showed impaired discrimination performance on the discrimination conditioning trials, reductions in early pretest CS-alone responses, and reductions in resting HR. However, on the non-drug test trials discrimination performance was normal in all cocaine groups. The results established that in spite of major changes in HR dynamics, learning of the HR discrimination was not affected by cocaine but that cocaine did interfere with the performance of the discrimination. Except for the highest 30 mg group, the performance decrement appeared to be related to a cocaine-produced reduction in the capacity to inhibit bradycardia responding to the safe CS-. It was suggested that this loss of inhibitory control may have been due to cocaine changes in a corticothalamic pathway that controls inhibition of bradycardia to a safe CS-.

The acute respiratory distress syndrome: definitions, severity and clinical outcome. An analysis of 101 clinical investigations.

OBJECTIVE: To determine possible changes in outcome from acute respiratory distress syndrome (ARDS) and to compare severity of lung injury and methods of treatment from 1967 to 1994. DATA SOURCES: Computerized (Medline, Current Contents) and manual (Cumulated Index Medicus) literature search using the key word and/or title ARDS. STUDY SELECTION: Only clinical studies published as full papers reporting data on both patient mortality (survival) and oxygenation index (PaO2/FIO2) were included. Single case reports, abstracts, reviews and editorials were excluded from evaluation. DATA EXTRACTION: Relevant data were extracted in duplicate, followed by quality checks on approximately 80% of data extracted. DATA SYNTHESIS: 101 papers reporting on 3264 patients were included: 48 studies (2207 patients) were performed in the USA, 43 studies (742 patients) in Europe and 10 studies (315 patients) elsewhere. Mortality reported in these studies was 53 +/- 22% (mean +/- SD), with no apparent trend towards a higher survival (1994: 22 studies, mortality 51 +/- 19%). The mean PaO2/FIO2 ratio remained unchanged throughout the observation period (118 +/- 47 mmHg). No correlation could be established between outcome and PaO2/FIO2 or lung injury score. Patients who underwent pressure-limited ventilation had a significantly lower mortality (35 +/- 20%) than patients on volume-cycled ventilation (54 +/- 22%) or patients for whom there was no precise information on ventilatory support (59 +/- 19%). Significantly lower PaO2/FIO2 ratios (61 +/- 17 mmHg) were observed in patients prior to extracorporeal lung assist, together with mortality rates in the range of those for conventionally treated patients (55 +/- 22%). CONCLUSIONS: The mortality of ARDS patients remained constant throughout the period studied. Therefore, the standard for outcome in ARDS should be a mortality in the 50% range. Neither PaO2/FIO2 ratio nor lung injury score was a reliable predictor for outcome in ARDS. Patients might benefit from pressure-limited ventilatory support, as well as extracorporeal lung assist. Since crucial data were missing in most clinical studies, thus preventing direct comparison, we emphasize the importance of using standardized definitions and study entry criteria.

Influence of positive airway pressure on the pressure gradient for venous return in humans.

To study the effect of positive airway pressure (Paw) on the pressure gradient for venous return [the difference between mean systemic filling pressure (Pms) and right atrial pressure (Pra)], we investigated 10 patients during general anesthesia for implantation of defibrillator devices. Paw was varied under apnea from 0 to 15 cmH(2)O, which increased Pra from 7.3 +/- 3.1 to 10.0 +/- 2.3 mmHg and decreased left ventricular stroke volume by 23 +/- 22%. Episodes of ventricular fibrillation, induced for defibrillator testing, were performed during 0- and 15-cmH(2)O Paw to measure Pms (value of Pra 7.5 s after onset of circulatory arrest). Positive Paw increased Pms from 10.2 +/- 3.5 to 12.7 +/- 3.2 mmHg, and thus the pressure gradient for venous return (Pms - Pra) remained unchanged. Echocardiography did not reveal signs of vascular collapse of the inferior and superior vena cava due to lung expansion. In conclusion, we demonstrated that positive Paw equally increases Pra and Pms in humans and alters venous return without changes in the pressure gradient (Pms - Pra).

Nalbuphine by PCA-pump for analgesia following hysterectomy: bolus application versus continuous infusion with bolus application.

The analgesic properties of the partial agonist-antagonist nalbuphine in the postoperative period are well known. When used for patient-controlled analgesia (PCA) the effectiveness of this substance is comparable to that of morphine or tramadol. However, the optimal programme for administration of nalbuphine in PCA-pumps has not been investigated. In particular, the combination of bolus administration vs bolus administration plus continuous basal administration is disputable. We hypothesized that the administration of an extra basal rate of nalbuphine in addition to the patient- triggered bolus administration and supplemental doses of diclofenac when required, would lead to a significant improvement in analgesia, without affecting the differences in vital signs and side effects. After approvement by the institutional ethics committee, 50 female patients (ASA I or II) scheduled for elective hysterectomy were included in a prospective, single-blinded study and randomized either into bolus-continuous (BC-)group (3 mg base rate/h, 1 mg bolus, 20 min lock out) or bolus (B-)group (no base rate, 1 mg bolus, 10 min lock out). During the observation period (up to 24 h postoperative) vital parameters, extent of analgesia (10-step VAS), and vigilance (5-step scale) were registered. Groups were compared by using unpaired Student t-test. A p<0.05 was considered to be significant. No differences were found in demographic data or vital parameters (MAP, PaO2, PaCO2, respiratory rate, heart rate, peripheral SaO2) during the observation period. Vital parameters showed no pathological changes in any group. With an identical rate of requirement for diclofenac (32 and 36%), analgesia in BC-group showed a decrease in VAS from 4.28+/-2.11 to 2.04+/-1.21 and from 3.64+/-2.20 to 2.08+/-0.96 in B-group. Vigilance was only marginally diminished in both groups. No serious side effects were found in either group. The consumption of nalbuphine (mg) was significantly higher in BC-group (70.28+/-13.85 vs. 47.44+/-22.99;p =0.0002) when compared to B-group. Subjective rating of effectiveness by the patients was similar in both groups. The two administration settings of nalbuphine by PCA pump have shown to be equally effective in the treatment of postoperative pain following hysterectomy. However, as the total amount of nalbuphine was significantly lower in B-group, the use of this administration schedule should be encouraged.

Opposing heart rate reactions associated with behavioral states of excitation and inhibition.

Following the development of an excitatory bradycardia conditioned response (CR) to a CS+ paired with a shock US on Day 1, three groups of rats were given one of three inhibitory training procedures on Day 2 with a different CS (CS-). Then on Day 3 the inhibitory capacity of each CS- was examined on a modified combined cue test in which CS- was given slightly before CS+ and on a reversal conditioning test in which CS- was now paired with the US. The three inhibitory procedures consisted of CS- alone (CSA) trials, explicitly unpaired (EUP) CS- and US trials, and truly random (TR) CS- and US trials. During inhibitory training, the bradycardia CR to CS+ was replaced with a tachycardia reaction to CS- in the EUP group but not in the other groups. Subsequent to inhibitory training the EUP group and to some extent also the TR group showed a decrement in the excitatory bradycardia response to CS+ when compared to the robust HR slowdowns displayed by the CSA group. It was suggested, within the context of opponent process theory, that the separate USs given the EUP and TR groups (especially the former) during inhibitory training may have led to conditioning of inhibitory tendencies to CS- and that these in turn generalized and decremented previous established bradycardia responding to CS+ and the development of a new bradycardia to CS- during reversal conditioning.

Baroreceptor involvement in classically conditioned heart rate responses of restrained rats.

A group (N = 8) of restrained, baroreceptor denervated rats and a sham-operated-control group (n = 8) received discriminated classical conditioning consisting of 30 reinforced trials in which a CS+ was paired with an electric shock US and 30 non-reinforced trials in which a different CS (CS-) was presented alone. The control group displayed a decelerative heart rate CR and a biphasic pressor-depressor blood pressure CR. The denervated group failed to show a heart rate CR but did show a pressor-only blood pressure CR. The URs of the denervated group consisted of a major depressor change in blood pressure and a slight tachycardia whereas the URs of the control group consisted of a slight pressor response and tachycardia. The results indicated that centrally initiated activity in the efferent vagal pathways mediating the decelerative HR CR in rats may be blocked by the absence of normal afferent baroreceptor neural discharge. An integrating role of baroreceptor input was also suggested for the URs.

The influence of ethanol on learned and reflexive heart rate responses of rats during classical aversive conditioning.

An unconditioned response was produced in rats by electric shock and resulted in a biphasic deceleration-acceleration of heart rate after 2.4 g of ethanol per kg. A monophasic acceleration occurred with saline and 0.8 g of ethanol per kg.

Adaptation and validation of a radioimmunoassay kit for measuring plasma cortisol in turbot.

Levels of cortisol in fish blood provide quantitative information on the degree of stress induced by a variety of stressors. It is also useful in describing the social status of individual fish within groups. The commercial production of radioimmunoassay (RIA) kits, such as the DPC Coat-A-Count radioimmunoassay kit, has considerably reduced the effort required for cortisol measurement. These kits employ human plasma based cortisol standards which are not compatible for use with non mammalian species such as fish e.g. turbot, Scophthalmus maximus (Rafinesque), blood due to the interference effect of lipids and steroid binding proteins present in the plasma. In this study the DPC kit was used following the removal of these lipids and steroid binding proteins from the plasma using an ethanol-hexane extraction. Excessive variability in the cortisol values obtained using this method deemed it unsatisfactory in overcoming the problem of incompatibility. A second modification of this technique that was tested involved the preparation of turbot specific standards for use in the preparation of modified standard curves. Using this method, an accuracy of 93.4% was achieved, as opposed to 79.6% using the kit human plasma based standards, and 47.1% using samples following lipid removal using an ethanol-hexane extraction. Based on analysis of accuracy, precision and reproducibility it is concluded that commercially available cortisol kits are suitable for use with turbot plasma, but a number of minor modifications are necessary.

Group process in teaching family dynamics to family practice residents.

Behavioral science training in family practice at Madigan Army Medical Center, Tacoma, Washington, has used small group seminars to teach family dynamics on an experiential level. The group process is similar enough to dynamics within families to facilitate understanding by introspection. Small groups are an efficient method for teaching, and allow experiential learning to occur spontaneously as well as on a planned basis.