Development of polymorphic microsatellites for Sillago sihama based on next-generation sequencing and transferability to Sillago japonica.

Sillago sihama (Forsskål, 1775), a commercially important marine fishery species in the Indo-West Pacific, is being developed as a target species for aquaculture and stock enhancement in China. However, due to the limitations of traditional isolation methods, the available microsatellite loci, or simple sequence repeats (SSRs), of S. sihama lack diversity. We used a stepwise approach including Illumina sequencing, primer screening, and SSR marker validation to develop diverse SSRs for S. sihama. A total of 853.48 Mb clean sequences were assembled with high coverage and sequencing depth, and 27,288 potential SSRs were identified. A set of 18 novel SSR markers with four type motifs including 11 di-, 1 tri-, 5 tetra-, and 1 hexanucleotide repeats were successfully isolated. The ranges of number of alleles per locus and observed and expected heterozygosities were 5-24, 0.226-0.968, and 0.319-0.950, respectively. The diversity parameters exhibited high levels of polymorphism in these 18 loci. Three loci with the presence of both null alleles and inbreeding showed significant deviation from Hardy-Weinberg equilibrium after Bonferroni correction. Moreover, 13 loci developed in S. sihama showed high transferability to the closely related species Sillago japonica. The polymorphic SSR markers developed in this study may serve as valuable tools for further basic and applied research on the genetic resources of S. sihama as well as S. japonica. Our results indicate that this approach, based on next-generation sequencing technology, is convenient, cost-effective, and suitable for SSR marker isolation in other sillaginid fishes.

Isolation and identification of the immune-relevant ribosomal protein L10 (RPL10/QM-like gene) from the large yellow croaker Pseudosciaena crocea (Pisces: Sciaenidae).

In order to investigate the immune role of ribosomal protein L10 (RPL10/QM-like gene) in marine fish, we challenged the large yellow croaker Pseudosciaena (= Larimichthys) crocea, the most important marine fish culture species in China, by injection with a mixture of the bacteria Vibrio harveyi and V. parahaemolyticus (3:1 in volume). Microarray analysis and real-time PCR were performed 24 and 48 h post-challenge to isolate and identify the QM-like gene from the gill P. crocea (designated PcQM). The expression level of the PcQM gene did not changed significantly at 24 h post-challenge, but was significantly downregulated at 48 h post-challenge, suggesting that the gene had an immune-modulatory effect in P. crocea. Full-length PcQM cDNA and genomic sequences were obtained by rapid amplification of cDNA ends (RACE)-PCR. The sequence of the PcQM gene clustered together with those of other QM-like genes from other aquatic organisms, indicating that the QM-like gene is highly conserved in teleosts.

Molecular characterization and structure analysis of RPL10/QM-like protein from the red drum Sciaenops ocellatus (Sciaenidae).

The QM-like gene encodes a ribosomal protein L10. Besides housekeeping roles in protein synthesis, QM-like proteins have multiple extraribosomal functions during cell growth, cell differentiation and apoptosis. We obtained the full-length cDNA of QM-like protein (designated as SoQM) from the salt water game fish Sciaenops ocellatus, using RACE-PCR. The sequence consists of 740 bp, encoding 215-amino acid residues with 24.60 kDa. The AA sequence of the SoQM protein contains a series of functional motifs that belong to the QM family signature, which is conserved among different species. The SoQM gene contains five introns and six exons. The expression pattern of SoQM as determined by RT-PCR indicated that SoQM mRNA was expressed in all tissues tested, including brain, gill, head-kidney, intestine, stomach, heart, spleen, blood, muscle, and gonads. The phylogenetic tree constructed with MEGA 4.0 showed that SoQM clusters together with that of other fish. It was found that the sequences of the SoQM gene are highly conserved, suggesting the fundamental and critical functions of SoQM in S. ocellatus. The three-dimensional structure of the SoQM protein core domain (4~169) was predicted by the Swiss-Model program. Compared with QM proteins in other species, the main structure of SoQM protein was conserved, while the C-terminal domain was different from other QM-like proteins. Prediction of the three-dimensional structure of SoQM would provide valuable insight into the molecular basis of protein function, allowing an effective design of experiments, such as site-directed mutagenesis, studies of disease-related mutations or structure-based design of specific inhibitors.

The analgesic and respiratory depressant actions of metorphamide in mice and rabbits.

Metorphamide (MET) elicited a potent, dose-dependent analgesia and respiratory depression in mice and rabbits. MET induced-analgesia was naloxone reversible and potentiated by bestatin. Naloxonazine, a relatively selective mu 1 blocker, at certain dosage (50 micrograms per rabbit, icv), could abolish the analgesia but not the respiratory inhibition produced by MET. Our result indicates that mu 1 receptors mediate the MET induced-analgesia but not its respiratory effect. Since MET is a mu- and kappa-ligand with very low delta activity, the MET induced respiratory depression may be mediated by mu 2 or kappa binding sites.

[A clinical trial of treating asthma of moderate severity with beclomethasone dipropionate aerosol].

In order to investigate the efficacy of steroid inhalation in treating asthma of moderate severity, a single-blind, randomized short-term (3-4 weeks) trial was performed in 25 asthmatics uncontrolled by salbutamol inhalation, oral aminophylline and beta 2-agonist. 22 patients finished the trial. Among them, twelve received beclomethasone dipropionate 300 mcg/day and ten received placebo. There was significant improvement in asthmatic symptoms and pulmonary function (FEV1.0, V50, V25) in the group treated with steroid inhalation at the end of this trial, whereas no significant changes were observed in the placebo group. The results demonstrated that steroid inhalation could effectively control asthma of moderate severity.

[A study of measuring cardiac output in patients during mechanical ventilation with CO2 rebreathing method].

Measurement of cardiac output with CO2 rebreathing method and thermodilution method was carried out in seven COPD patients undergoing mechanical ventilation. Mixed venous PCO2 could be measured reasonably well with CO2 rebreathing method. There was a very significant positive correlation between the CO2 method and thermodilution method for measuring cardiac output (r = 0.9130, P < 0.01). The change of cardiac output before and after infusion of sodium nitroprusside could be reflected by CO2 rebreathing method. We conclude that the cardiac output in COPD patients undergoing mechanical ventilation could be measured reasonably well with CO2 rebreathing method.

[The effects of sodium nitroprusside and almitrine bismesylate on blood gases, hemodynamics and oxygen delivery in patients with cor pulmonale].

The effects of sodium nitroprusside and almitrine bismesylate on blood gases, hemodynamics and oxygen delivery were respectively studied in twenty-one and eighteen patients with cor pulmonale. The treatment of sodium nitroprusside resulted in significant decrease in pulmonary artery pressure. Although physiological shunt was increased (from 12.6 +/- 10.5 to 20.1 +/- 10.9%, P < 0.01) and arterial oxygen tension was decreased (from 12.6 +/- 4.0 to 9.9 +/- 2.5 kPa, P < 0.01) in patients undergoing mechanical ventilation, cardiac index (from 44.8 +/- 10.5 to 53.3 +/- 12.8 ml.s-1/m-2, P < 0.05) and oxygen delivery index (from 7.8 +/- 1.7 to 9.2 +/- 2.3 ml.s-1/m-2, P < 0.05) were improved. After treatment with almitrine bismesylate, improvement of arterial oxygen tension was observed in patients with spontaneous breathing (from 6.7 +/- 0.6 to 7.8 +/- 0.6 kPa, P < 0.05) and undergoing mechanical ventilation (from 10.9 +/- 1.9 to 13.4 +/- 2.5 kPa, P < 0.01), but increment of mean pulmonary artery pressure (from 3.8 to 0.5 and 3.1 +/- 0.8 to 6.3 +/- 0.7 and 3.6 +/- 0.9 kPa, respectively, P < 0.01) was noted.

[A comparison between nasotracheal and orotracheal intubation in treating chronic obstructive pulmonary disease complicated by respiratory failure].

Endotracheal intubation and mechanical ventilation were given to 100 COPD patients complicated by respiratory failure from Dec. 1977 to May 1990. Among them 42 were intubated through mouth. The tubes were kept from 1 to 6 days. In only 8 cases (19.0%) the tubes could be withdrawn according to the criterion, and 25 patients (59.0%) were tracheostomized. Nasotracheal intubation were given to 58 patients. The tubes were kept from 1 to 220 days and could be withdrawn in 39 cases (81.0%), with an incidence higher than that in patients intubated orotracheally (P < 0.01). Tracheostomy mas given to 6 patients intubated nasotracheally (10.3%), being fewer than in patients intubated through mouth (P < 0.01). Although the tubes have been kept for longer time, the duration of hospitalization was not prolonged.

[Effects of external diaphragm pacing on transdiaphragmatic pressure, ventilation and arterial blood gases in healthy volunteers].

The effects of external diaphragm pacing (EDP) were studied in seven healthy volunteers. During EDP the movement of both left and right hemidiaphragms increased on average 1.28 and 1.30 cm respectively. The transdiaphragmatic pressure also increased from 8.63 +/- 1.576 cm H2O to 15.18 +/- 1.946 cm H2O (P less than 0.01). Inductive plethysmography showed that with EDP the mean inspiratory flow rate increased from 308 +/- 28.6 ml/sec. to 454 +/- 36.6 ml/sec. (P less than 0.01), but there was no change in respiratory rate, inspiratory time and the ratio of inspiratory time to respiratory cycle. Both tidal volume and minute volume of ventilation increased from 419 +/- 33.9 ml to 691 +/- 71.5 ml (P less than 0.01) and from 7.02 +/- 0.74 l/min. to 10.14 +/- 0.73 l/min. (P less than 0.01) respectively. Accompanied with the change of ventilation, the consumption of oxygen and the production of CO2 also increased from 258 +/- 14.9 ml/min. to 310 +/- 15.0 ml/min. (P less than 0.05) and from 228 +/- 11.4 ml/min. to 299 +/- 25.9 ml/min. (P less than 0.05) respectively. Consequently PaCO2 reduced from 5.24 +/- 0.22 kPa to 4.27 +/- 0.25 kPa (P less than 0.05), whereas PaO2 increased from 12.7 +/- 0.32 kPa to 14.5 +/- 0.42 kPa (P less than 0.01).

[The effects of almitrine bismesylate in chronic obstructive pulmonary disease with CO2 retention].

Ten cases of chronic obstructive pulmonary disease (COPD) were examined before and 2 hours after oral administration of 150 mg of almitrine bismesylate while breathing 31% oxygen. The agent induced a rise in mouth occlusion pressure (P 0.1) from 2.67 +/- 0.64 cmH2O to 3.74 +/- 1.02 cmH2O (P less than 0.01). However the central inspiratory drive and ventilation response to hypercapnia did not change after taking almitrine. VT/Ti increased from 0.32 +/- 0.06 L/sec. to 0.40 +/- 0.08 L/sec. (P less than 0.01) and RR, Ti, Ti/Ttot were not changed, so VT and VE increased from 0.37 +/- 0.10 L to 0.42 +/- 0.16 L (P less than 0.05) and from 7.70 +/- 1.72 L to 8.85 +/- 2.28 L (P less than 0.05) respectively. VA also increased from 3.34 +/- 0.06 L to 4.14 +/- 0.86 L (P less than 0.01). Analysis of blood gases showed a marked increase in PaO2 from 88.70 +/- 16.19 mmHg to 109.10 +/- 25.57 mmHg (P less than 0.05) and a decrease in PaCO2 from 61.73 +/- 12.15 mmHg to 54.01 +/- 10.37 mmHg (P less than 0.05). Our results suggested that almitrine bismesylate could improve the blood gases in COPD patients with CO2 retention as it is a respiratory stimulant which might act through a peripheral mechanism.

Non-uniform lung elastic properties and the slope of the alveolar plateau.

Dispersion of elastic properties within the lungs may contribute to the creation of a sloping alveolar plateau of the single-breath N2 washout curve. In this study we have systematically explored the behaviour of a simple two-compartment lung model with non-uniform pressure-volume characteristics. The inhomogeneity was expressed in terms of the ratio between the shape constants (k1/k2) of the compartmental pressure-volume curves and the ratio between compartmental RV/TLC (R-RV/TLC). The model simulations indicate that normal slope magnitudes can be achieved with a moderate degree of inhomogeneity (k1/k2 and R-RV/TLV = 2). The model simulations also show that a phase II-like shape of the curve--of non dead space origin--may occur and furthermore that single-breath TLC determinations increasingly underestimate true TLC as the R-RV/TLC deviates from unity. For a given degree of inhomogeneity, the model predicts the slope to increase with overall RV/TLC and k but decrease with TLC. The relations between these overall factors and the slope of phase III are also found in a patient group, where these factors explain approximately 61% of the slope variance.

Combined surfactant therapy and inhaled nitric oxide in rabbits with oleic acid-induced acute respiratory distress syndrome.

Intratracheal administration of surfactant and inhaled nitric oxide (INO) have had variable effects in clinical trials on patients with acute respiratory distress syndrome (ARDS). We hypothesized that combined treatment with exogenous surfactant and INO may have effects in experimental ARDS. After intravenous infusion of oleic acid in adult rabbits and 4-6 h of ventilation, there was more than a 40% reduction in both dynamic compliance (Cdyn) of the respiratory system and functional residual capacity (FRC), a 50% increment of respiratory resistance (Rrs), a 70% reduction in PaO2 /FIO2, and an increase in intrapulmonary shunting (Q S/Q T) from 4.4 to 33.5%. The animals were then allocated to groups receiving (1) neither surfactant nor INO (control), (2) 100 mg/kg of surfactant (S) administered intratracheally, (3) 20 ppm INO (NO), or (4) 100 mg/kg of surfactant and 20 ppm INO (SNO), and subsequently ventilated for 6 h. After the period of ventilation, the animal lungs were used for analysis of disaturated phosphatidylcholine (DSPC) and total proteins (TP) in bronchoalveolar lavage fluid (BALF), and for determination of alveolar volume density (VV). The animals in the control group had the lowest survival rate, and no improvement in lung mechanics and blood oxygenation, whereas those in the S group had a modest but statistically significant improvement in Cdyn, Rrs, PaO2 and FRC, reduced Q S/Q T, lowered minimum surface tension (gammamin) of BALF, and increased DSPC/ TP and alveolar VV. The NO group had increased PaO2 and reduced Q S/Q T. The SNO group showed improved Cdyn, Rrs, FRC, DSPC/TP, alveolar VV, and gammamin of BALF comparable to the S group, but there was a further increase in survival rate and PaO2, and additional reduction in Q S/Q T and TP in BALF. These results indicate that, in this animal model of ARDS, a combination of surfactant therapy and INO is more effective than either treatment alone.