The use of gene clusters to infer functional coupling

Previously, we presented evidence that it is possible to predict functional coupling between genes based on conservation of gene clusters between genomes. With the rapid increase in the availability of prokaryotic sequence data, it has become possible to verify and apply the technique. In this paper, we extend our characterization of the parameters that determine the utility of the approach, and we generalize the approach in a way that supports detection of common classes of functionally coupled genes (e.g., transport and signal transduction clusters). Now that the analysis includes over 30 complete or nearly complete genomes, it has become clear that this approach will play a significant role in supporting efforts to assign functionality to the remaining uncharacterized genes in sequenced genomes.     

耐力训练过程中大鼠体内糖储备及胰岛激素变化与黄芪、生脉穴位注射的干预效应

目的:穴位注射疗法在临床应用较多,但在运动医学领域研究不多。观察穴位注射黄芪、生脉对耐力训练大鼠糖储备和运动能力的影响。方法:实验于2004-07在陕西师范大学完成。①实验分组:健康雄性SD大鼠32只,体质量180～220g,随机抽签法分为安静对照组、训练对照组、生理盐水组、药物注射组,每组8只。②实验方法:建立穴位注射黄芪、生脉大鼠的耐力跑台训练实验模型,安静对照组安静笼饲养。训练对照组、生理盐水组、药物注射组先于动物跑台上进行5周适应性训练,之后跑速每周递增,5d/周,共5周;然后进行2周的大强度耐力训练,30min/d,7d/周,共2周。训练对照组、生理盐水组、药物注射组第8周第1天以速度为35m/min运动至力竭。③实验评估:7周后取材测定肝糖原、肌糖原、血清胰岛素、胰高血糖素的变化。实验中对动物处置符合动物伦理学标准。结果:纳入大鼠32只,均进入结果分析。①通过大强度耐力训练,药物注射组与其他3组相比,肝糖原含量均升高(P<0.05);训练对照组肌糖原比安静对照组降低(P<0.05),生理盐水组与训练对照组相比则显著性升高(P<0.01)。②训练对照组胰岛素比安静对照组明显降低(P<0.01);生理盐水组及药物注射组都能抑制这种降低的趋势(P<0.01);药物注射组胰高血糖素较安静对照组、训练对照组要高,且有显著性差异(P<0.01)。结论:穴位注射黄芪、生脉使大强度耐力训练大鼠体内糖储备显著增加,同时可以提高胰岛激素水平,从而提高了大鼠的运动能力。     

Blood usage in lung transplantation

BACKGROUND: Few published data are available regarding perioperative blood usage in lung transplantation. STUDY DESIGN AND METHODS: The medical records of all patients undergoing lung transplantation at a university medical center in 1994 and 1995 were reviewed. RESULTS: Ninety patients underwent lung transplantation during this period. Six patients were excluded: two received a living related-donor lung, three underwent retransplantation and one underwent concomitant repair of a tetralogy of Fallot. Of the 84 evaluable patients, 59 underwent single lung transplantation and 25 double lung transplantation. Double-lung recipients used more red cells (6.4 vs. 1.7 units, p = 0.0002) and were more likely to receive red cells, platelets, plasma, or any component (92 vs. 32%, p< or =0.0001) than were single-lung recipients. Double- lung recipients were more likely to require cardiopulmonary bypass (40 vs. 12%, p = 0.003), and cardiopulmonary bypass was associated with greater transfusion requirements (p< or =0.0001). However, among patients requiring cardiopulmonary bypass, blood use did not differ between those undergoing double lung transplantation and those undergoing single lung transplantation. In the subset of patients not requiring cardiopulmonary bypass, double-lung recipients received more red cells (4.5 vs. 0.7 units, p< or =0.0001) and more plasma (2.0 vs. 0.2 units, p = 0.006). CONCLUSION: Double-lung recipients require more perioperative transfusions than single-lung recipients. The greater transfusion requirement is due to the more frequent need for cardiopulmonary bypass as well as the greater complexity of the procedure. These data are useful for developing surgical blood ordering guidelines for lung transplantation.     

Flavocoxid,a dual inhibitor of cyclooxygenase and 5-lipoxygenase,blunts pro-inflammatory phenotype activation in endotoxin-stimulated macrophages

Background and purpose:

The flavonoids, baicalin and catechin, from Scutellaria baicalensis and Acacia catechu, respectively, have been used for various clinical applications. Flavocoxid is a mixed extract containing baicalin and catechin, and acts as a dual inhibitor of cyclooxygenase (COX) and 5-lipoxygenase (LOX) enzymes. The anti-inflammatory activity, measured by protein and gene expression of inflammatory markers, of flavocoxid in rat peritoneal macrophages stimulated with Salmonella enteritidis lipopolysaccharide (LPS) was investigated.

Experimental approach:

LPS-stimulated (1 µg·mL⁻¹) peritoneal rat macrophages were co-incubated with different concentrations of flavocoxid (32–128 µg·mL⁻¹) or RPMI medium for different incubation times. Inducible COX-2, 5-LOX, inducible nitric oxide synthase (iNOS) and inhibitory protein κB-α (IκB-α) levels were evaluated by Western blot analysis. Nuclear factor κB (NF-κB) binding activity was investigated by electrophoretic mobility shift assay. Tumour necrosis factor-α (TNF-α) gene and protein expression were measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay respectively. Finally, malondialdehyde (MDA) and nitrite levels in macrophage supernatants were evaluated.

Key results:

LPS stimulation induced a pro-inflammatory phenotype in rat peritoneal macrophages. Flavocoxid (128 µg·mL⁻¹) significantly inhibited COX-2 (LPS = 18 ± 2.1; flavocoxid = 3.8 ± 0.9 integrated intensity), 5-LOX (LPS = 20 ± 3.8; flavocoxid = 3.1 ± 0.8 integrated intensity) and iNOS expression (LPS = 15 ± 1.1; flavocoxid = 4.1 ± 0.4 integrated intensity), but did not modify COX-1 expression. PGE₂ and LTB₄ levels in culture supernatants were consequently decreased. Flavocoxid also prevented the loss of IκB-α protein (LPS = 1.9 ± 0.2; flavocoxid = 7.2 ± 1.6 integrated intensity), blunted increased NF-κB binding activity (LPS = 9.2 ± 2; flavocoxid = 2.4 ± 0.7 integrated intensity) and the enhanced TNF-α mRNA levels (LPS = 8 ± 0.9; flavocoxid = 1.9 ± 0.8 n-fold/β-actin) induced by LPS. Finally, flavocoxid decreased MDA, TNF and nitrite levels from LPS-stimulated macrophages.

Conclusion and implications:

Flavocoxid might be useful as a potential anti-inflammatory agent, acting at the level of gene and protein expression.     

Role of Endothelium/Nitric Oxide and Cyclic AMP in Isoproterenol Potentiation of 17ß-Estradiol-Mediated Vasorelaxation

Background: Calcitonin gene-related peptide (CGRP) is known to have an extremely potent and prolonged vasodilator effect on the coronary arteries. Studies have shown that CGRP increased coronary blood flow and alleviated reperfusion injury in vitro. It is still unknown, however, whether exogenous CGRP has a protective effect on the reperfusion heart associated with cardiopulmonary bypass (CPB). Methods: An in vivo porcine model of CPB was established. Twenty pigs, 10 controls and 10 CGRP used animals (CGRP group), were performed a median sternotomy followed by a standard CPB. All the hearts were arrested for 45 minutes. In the CGRP group, 1mg/kg CGRP was added into the cardioplegia, and another 1mg/kg was reperfused just before the aortic cross-clamp was removed. In both groups, myocardial microvascular perfusion, coronary arterial microvessel diameter and microvessel blood flow were detected by a laser doppler flowmeter and a contact microscope with TV monitor on five consecutive time perioperatively. Result: Myocardial microvascular perfusion was significantly higher and coronary arterial microvessel diameter was larger in the CGRP group on every point of time of reperfusion compared to those in the control group. In the CGRP group, microvessel blood flow also improved significantly than that in the control group during reperfusion. Conclusion: CGRP improves myocardial microcirculation during cardiac ischemia-reperfusion associated with CPB and could become a new, potent myocardial protector.     

Prospective randomised comparison of single-dose versus multiple-dose cefuroxime for prophylaxis in coronary artery bypass grafting

To assess the efficacy of single-dose antibiotic prophylaxis in coronary artery bypass grafting, 1,016 consecutive patients were prospectively randomized to receive either a single dose or a three-day course of cefuroxime. Nine patients (0.9 %) died within seven days; no death was caused by infection. For various reasons 163 other patients were not evaluable. Therefore, 844 patients were evaluated. Patients in group A (n=419) received 20 mg/kg cefuroxime intravenously at induction of anaesthesia; group B (n=425) received the same dose followed by 750 mg t.i.d. for three consecutive days. Both groups were comparable regarding all risk factors. The efficacy of the prophylactic regimens was evaluated by comparison of occurrence of wound infection in both groups. No significant differences in wound infection were observed between the two treatment groups: sternal site infection in the single-dose prophylaxis group was 14 % versus 13 % in the three-day course group; donor site infection occurred in 38 % versus 39 %. It is concluded that in coronary artery bypass grafting a single dose of cefuroxime is as effective as a three-day course in the prevention of wound infection.     

Photoreceptor degeneration induced by the expression of simian virus 40 large tumor antigen in the retina of transgenic mice.

Expression of the viral oncogene encoding the simian virus 40 (SV40) large tumor antigen (T antigen) typically promotes tumorigenesis in mammalian cells. To generate transgenic mice that express T antigen in rod photoreceptors, a chimeric construct consisting of a mouse opsin promoter fragment fused to the coding region of SV40 T antigen was generated. Expression of T antigen in the transgenic retina began at early stages of postnatal development concomitant with expression of endogenous opsin. Instead of inducing hyperplasia or tumor formation, T-antigen expression caused a rapidly progressing photoreceptor degeneration. The degeneration was accompanied by sustained DNA synthesis in photoreceptor cells, as evidenced by incorporation of [3H]thymidine and by the appearance of mitotic figures at postnatal day 10, a stage when nontransgenic photoreceptor cells are postmitotic and quiescent. Although transgenic photoreceptor cells undergo S phase and enter mitosis, the consequences of T-antigen expression are not proliferation and tumorigenesis but proliferation and cell death.     

Most Patients with Colorectal Tumors at Young Age Do Not Visit a Cancer Genetics Clinic

Purpose  This study examined the referral process for genetic counseling at a cancer genetics clinic in patients with colorectal cancer and to search for determinants of variation in this referral process. Methods  Patients who were recently diagnosed with colorectal cancer at a young age or multiple cancers associated with Lynch syndrome, hereditary nonpolyposis colorectal cancer, (N = 119) were selected from PALGA, the nationwide network and registry of histopathology and cytopathology in the Netherlands. In a retrospective analysis, we examined whether these patients visited a cancer genetics clinic and identified determinants for referral to such a clinic. Factors of patients, professional practice, and hospital setting were explored with logistic regression modeling. Results  Thirty-six (30 percent) patients visited a cancer genetics clinic. Seventy percent of patients whom the surgeon referred to a cancer genetics clinic decided to visit such a clinic. Analysis of determinants showed that patients with whom the surgeon discussed referral and that were treated in a teaching hospital were more likely to visit a cancer genetics clinic. Conclusion  The referral process is not optimally carried out. To deliver optimal care for patients suspected of hereditary colorectal cancer, this process must be improved with interventions focusing on patient referral by surgeons and raising awareness in nonteaching hospitals. This work was supported by ZonMw, the Netherlands Organization for Health Research and Development.     

Ultra-protective tidal volume: how low should we go?

Applying tidal volumes of less than 6 mL/kg might improve lung protection in patients with acute respiratory distress syndrome. In a recent article, Retamal and colleagues showed that such a reduction is feasible with conventional mechanical ventilation and leads to less tidal recruitment and overdistension without causing carbon dioxide retention or auto-positive end-expiratory pressure. However, whether the compensatory increase in the respiratory rate blunts the lung protection remains unestablished.Further reducing tidal volumes beyond the standard 6 mL/kg is an appealing goal in patients with acute respiratory distress syndrome (ARDS) [1]. Such reduction could decrease the tidal stretch imposed on the lung, potentially attenuating further the ventilator-induced lung injury [2]. In fact, tidal volumes of less than 6.5 mL/kg and as low as 4 mL/kg were recently associated with increased survival in patients with ARDS [3]. One of the main obstacles to such a strategy is the potential for carbon dioxide (CO₂) retention and severe acidosis. To avoid this, specialized techniques, such as high-frequency oscillatory ventilation and extracorporeal CO₂ removal, have been previously tested with mixed results [4-6].In the previous issue of Critical Care, Retamal and colleagues proposed that lower tidal volumes could be used with conventional positive-pressure ventilation without leading to CO₂ retention [1]. A reduction in tidal volume from 6 to 4 mL/kg was feasible with a decrease in the instrumental dead space and an increase in the respiratory rate. In patients with ARDS, the dead space is a marker of disease severity [7]. Consequently, very low tidal volumes can be difficult to use in practice, especially in very sick patients, because the necessary increase in respiratory rate might cause significant auto-positive end-expiratory pressure (auto-PEEP). Luckily, patients with severe ARDS also tend to have low lung compliance [8], making their lungs inflate and deflate fast. Therefore, this restrictive ventilatory pattern allows the safe use of high respiratory rates without leading to significant auto-PEEP.Retamal and colleagues [1] should be congratulated for their careful design of the ventilator protocol in the 4 mL/kg phase, which allowed an effective CO₂ elimination. The bottom line is that if one decides to use very low tidal volumes with high respiratory rates, attention to the details is invaluable. First, the removal of any dispensable dead space, including substituting an external heated humidifier by the heat-moisture exchanger, is imperative. Second, the use of volume-controlled ventilation helps to keep short inspiratory times. Peak airway pressures may increase, but the preserved expiratory time guarantees low auto-PEEP and, consequently, low plateau pressures. For safety, plateau pressures and auto-PEEP should be measured periodically. Third, in selected cases with high recruitability, the alveolar dead space can be minimized through recruitment maneuvers and higher PEEP values. Finally, the use of a short end-inspiratory pause is encouraged to improve the CO₂ elimination [9]. These measures will improve the safety and optimize the CO₂ elimination of a strategy with very low tidal volumes, even with higher-than-normal respiratory rates.However, even successfully avoiding CO₂ retention, this strategy has yet to be proven effective in terms of further lung protection. We believe that two aspects should be taken into consideration. The first is whether the strategy attenuated the mechanisms of lung injury. The authors performed computed tomography scans in all patients at tidal volumes of both 4 and 6 mL/kg and showed that the amount of cyclic recruitment-derecruitment and hyperinflation decreased after reducing the tidal volume. Although the absolute reduction was small (less than 1% of the lung weight), this finding is suggestive of decreased injury per breath. The second aspect is that an increased respiratory rate can be injurious per se [10]. It would be important to know whether the compensatory increase of the respiratory rate blunted the protective effect per breath of the tidal volume reduction.This tradeoff was emphasized recently in a model of the energy delivered by the ventilator as a surrogate for the potential lung damage [11]. Decreases in tidal volume require disproportionate increases in respiratory rate to maintain alveolar ventilation, and so more energy can be delivered to the lungs even at reduced stress and strain per breath. Though purely theoretical, this hypothesis helps reconcile our expectation of a further protective effect of very low tidal volumes with the recent findings of harmful or null effect of oscillatory high-frequency ventilation [5,6]. In these trials, it is possible that the reduction in lung injury per breath was offset by the very high respiratory rates applied.Finally, Retamal and colleagues [1] followed their patients for 5 to 30 minutes only. Since lower tidal volumes tend to promote atelectasis, especially under insufficient PEEP [12], a longer observation time perhaps would have shown an increase in atelectasis and driving pressures, opposing the benefits initially achieved.In conclusion, we are convinced that a strategy with very low tidal volumes (4 mL/kg) is feasible with conventional positive-pressure ventilation. This strategy could be used in patients with high plateau pressures or high driving pressures with standard 6 mL/kg tidal volumes, but we need more data in terms of lung protection before we can recommend this strategy to every patient with ARDS.