Whole-genome random sequencing and assembly of Haemophilus influenzae Rd.

An approach for genome analysis based on sequencing and assembly of unselected pieces of DNA from the whole chromosome has been applied to obtain the complete nucleotide sequence (1,830,137 base pairs) of the genome from the bacterium Haemophilus influenzae Rd. This approach eliminates the need for initial mapping efforts and is therefore applicable to the vast array of microbial species for which genome maps are unavailable. The H. influenzae Rd genome sequence (Genome Sequence DataBase accession number L42023) represents the only complete genome sequence from a free-living organism.

The minimal gene complement of Mycoplasma genitalium.

The complete nucleotide sequence (580,070 base pairs) of the Mycoplasma genitalium genome, the smallest known genome of any free-living organism, has been determined by whole-genome random sequencing and assembly. A total of only 470 predicted coding regions were identified that include genes required for DNA replication, transcription and translation, DNA repair, cellular transport, and energy metabolism. Comparison of this genome to that of Haemophilus influenzae suggests that differences in genome content are reflected as profound differences in physiology and metabolic capacity between these two organisms.

Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii.

The complete 1.66-megabase pair genome sequence of an autotrophic archaeon, Methanococcus jannaschii, and its 58- and 16-kilobase pair extrachromosomal elements have been determined by whole-genome random sequencing. A total of 1738 predicted protein-coding genes were identified; however, only a minority of these (38 percent) could be assigned a putative cellular role with high confidence. Although the majority of genes related to energy production, cell division, and metabolism in M. jannaschii are most similar to those found in Bacteria, most of the genes involved in transcription, translation, and replication in M. jannaschii are more similar to those found in Eukaryotes.

Does a history of cardiac disease or hypertension increase mortality following primary elective total hip arthroplasty?

Preoperative co-morbidities such as known coronary artery disease have commonly deemed a patient at 'high risk' for primary elective Total Hip Arthroplasty (THA). We prospectively collected data on 1744 patients who underwent primary elective THA between 1998 and 2004; 273 had a history of cardiac disease defined as a previous hospital admission with a diagnosis of angina pectoris or myocardial infarction; 594 patients had hypertension defined as that requiring treatment with antihypertensives. We also had data on preoperative age, sex and body mass index (BMI). There was no statistically significant increase in early mortality at three months with a history of cardiac disease or hypertension and this remained so when adjusting for the other factors in a multivariate analysis. Sex or BMI also did not have a statistically significant effect on the risk of death within three months. Increasing age was the only significant risk factor for early mortality (p<0.001). Longer-term mortality at two and five years in relation to these factors was also examined. Statistical analysis revealed that coronary history now showed a highly significant association (p<0.001) with long-term mortality in patients who survived more than three months. This remained significant (p=0.002) when adjusted for the other factors. Hypertension continued to have no effect, as did BMI. Age remained a significant risk factor and ASA was also a predictor of death, as has been previously shown. The overall long-term mortality following THAwas less than expected from the normal population, even in the subgroup with a coronary history. This study will assist clinicians when advising patients who have one of these common risk factors when seeking primary elective THA.

Findings emerging from complete microbial genome sequences.

Sixteen microorganisms, including one eukaryote, four archaeons, and 11 eubacteria, have been completely sequenced and published. More than 50 genomes are scheduled to be completed by the year 2000. This explosive growth of information is forcing change in many scientific disciplines (e.g. bioinformatics and molecular genetics), spawning new fields, and even changing the way scientific information is used and shared. Novel, global genome sequence comparisons seem slow to appear but the infrastructure for these projects is being built, and we expect exciting developments in the near future.

Total knee replacement in morbidly obese patients. Results of a prospective, matched study.

The results of 41 consecutive total knee replacements performed on morbidly obese patients with a body mass index > 40 kg/m(2), were compared with a matched group of 41 similar procedures carried out in non-obese patients (body mass index < 30 kg/m(2)). The groups were matched for age, gender, diagnosis, type of prosthesis, laterality and pre-operative Knee Society Score. We prospectively followed up the patients for a mean of 38.5 months (6 to 66). No patients were lost to follow-up. At less than four years after operation, the results were worse in the morbidly obese group compared with the non-obese, as demonstrated by inferior Knee Society Scores (mean knee score 85.7 and 90.5 respectively, p = 0.08; mean function score 75.6 and 83.4, p = 0.01), a higher incidence of radiolucent lines on post-operative radiographs (29% and 7%, respectively, p = 0.02), a higher rate of complications (32% and 0%, respectively, p = 0.001) and inferior survivorship using revision and pain as end-points (72.3% and 97.6%, respectively, p = 0.02). Patients with a body mass index > 40 kg/m(2) should be advised to lose weight prior to total knee replacement and to maintain weight reduction. They should also be counselled regarding the inferior results which may occur if they do not lose weight before surgery.

Screening for herpesvirus genomes in common acute lymphoblastic leukemia.

There is epidemiological evidence that infection may play a role in the etiology of childhood leukemia in particular common B cell precursor acute lymphoblastic leukemia. A panel of 20 leukemic samples (panel 1) was examined for the presence of four lymphotropic herpesviruses using conventional molecular techniques. A second independent panel of 27 leukemic samples (panel 2), along with 28 control peripheral blood samples from children with other forms of cancer, was tested for the presence of the same four viruses using sensitive real-time quantitative PCR. While herpesvirus genomes were detected, they were present at very low levels; detection rates and levels were similar in the leukemic and control panels. In addition we surveyed 18 leukemic samples (five from panel 1, six from panel 2 and a further seven samples not previously analyzed) using a degenerate PCR assay capable of detecting the genomes of known herpesviruses plus putative new members of the family. No novel herpesvirus genomes were detected suggesting that a herpesvirus is unlikely to be etiologically involved as a transforming agent in common acute lymphoblastic leukemia.

Practical experience and confidence in managing emergencies among preregistration house officers.

OBJECTIVES: To assess the experience gained by pre-registration house officers (PRHOs) at the end of their first post. To assess confidence in managing common emergencies and experience gained in practical procedures. To compare traditional six month posts with four month posts and to compare the experiences of PRHOs posted in teaching hospitals (THs) with those based in district general hospitals (DGHs). DESIGN: Interview questionnaire. PARTICIPANTS: 152 graduates from Edinburgh University Medical School in 2000 who had completed their first PRHO post by February 2001. RESULTS: There were few significant differences in confidence in managing emergencies and in numbers of practical procedures attempted between respondents from four and six month posts or between those holding TH and DGH posts. PRHOs had gained little experience in practical procedures: fewer than 15% had performed five or more of a number of procedures including lumbar puncture, pleural aspiration, chest drainage, and insertion of nasogastric tube. A high proportion of PRHOs indicated that they felt confident initiating management of conditions in specialties of which they had little or no experience. CONCLUSIONS: Rotations of three four month posts do not seem to reduce overall experience in the PRHO year. There is little difference in experience gained between TH and DGH posts. PRHOs perform few practical procedures and some may be overconfident in their own abilities.

Evidence for 5-HT1-like receptor-mediated vasoconstriction in human pulmonary artery.

1. The 5-hydroxytryptamine (5-HT) receptors mediating contraction of human isolated pulmonary artery rings were investigated. Responses to the agonists 5-carboximidotryptamine (5-CT, non-selective 5-HT1 agonist), sumatriptan (5-HT1D-like receptor agonist), 5-HT and 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT, 5-HT1A receptor agonist) were studied. Responses to 5-HT and sumatriptan in the presence of the antagonists, methiothepin (non-selective 5-HT1+2-receptor antagonist), ketanserin (5-HT2A receptor antagonist) and the novel antagonist, GR55562 (5-HT1D receptor antagonist) were also studied. 2. All agonists contracted human pulmonary artery ring preparations in the following order of potency 5-CT > 5-HT = sumatriptan > 8-OH-DPAT. Maximum responses to 5-HT, 5-CT and sumatriptan were not significantly different. 3. Methiothepin 1 nM and 10 nM, but not 0.1 nM reduced the maximum contractile responses to 5-HT but did not alter tissue sensitivity to 5-HT. Methiothepin 0.1 nM, 1 nM and 10 nM had a similar effect on responses to sumatriptan. 4. The 5-HT2A receptor antagonist ketanserin (10 nM, 100 nM and 1 microM) also reduced the maximum contractile response to both 5-HT and sumatriptan without affecting tissue sensitivity to these agonists. 5. The novel 5-HT1D receptor antagonist, GR55562, inhibited responses to 5-HT and sumatriptan in a true competitive fashion. 6. The results suggest that the human pulmonary artery has a functional population of 5-HT1D-like receptors which are involved in the contractile response to 5-HT.

Bronchodilator and pre-protective effects of urodilatin in bovine bronchi in vitro: comparison with atrial natriuretic peptide.

1. This study examined the activity and mechanisms of action of urodilatin in bovine bronchi. For comparison, the ability of urodilatin to evoke bronchodilatation or protect against subsequent challenge was compared to that of the closely related peptide alpha-human atrial natriuretic peptide (ANP). 2. Urodilatin reversed methacholine-evoked contraction in a concentration-dependent manner in bovine bronchi. In the absence of any attempt to prevent degradation by neutral endopeptidases, urodilatin was more potent than ANP in this tissue. 3. The bronchodilator properties of urodilatin were significantly augmented by the neutral endopeptidase inhibitor, phosphoramidon (3.68 x 10(-5) M). This provides evidence for at least partial degradation of urodilatin by neutral endopeptidases. With phosphoramidon present, urodilatin and ANP were equipotent. 4. In the presence of phosphoramidon (3.68 x 10(-5) M), pre-incubation with urodilatin (10(-6) M) had a protective effect against subsequent methacholine-induced contraction. This action of urodilatin was quantitatively similar to that of ANP in the presence of this endopeptidase inhibitor. 5. The actions of urodilatin appear to involve ATP-sensitive K+ channels since tolbutamide (10(-6) - 10(-5) M) significantly attenuated the relaxations induced by this peptide. 6. Small conductance Ca(2+)-activated K+ channels seem likewise to be implicated in the actions of urodilatin since blockade of these channels with apamin (10(-7) - 10(-6) M) resulted in a marked attenuation of urodilatin-evoked responses. 7. The presence of charybdotoxin (10-9 M-10-M) had no significant effect on subsequent responses tourodilatin suggesting that large conductance Ca2+-activated K+ channels are not involved in the relaxations evoked by this peptide.8. In the presence of phosphoramidon (3.68 x 10-5 M), urodilatin (10-6 M) evoked elevation of cyclic GMP levels within bovine bronchial tissue. Levels of cyclic GMP increased significantly within 5-10 s in response to this peptide and preceded the initiation of relaxant responses. Maximum increases in cyclic GMP levels were reached within 5 min; the time required for maximal relaxation evoked by this peptide.9. In conclusion, urodilatin, like ANP reversed and protected against, subsequent methacholine-induced bronchoconstriction; an action enhanced by the presence of phosphoramidon (3.68 x 1O-5 M).Associated with these actions of urodilatin was a rise in cyclic GMP levels as well as the opening of ATP-sensitive K+ and small conductance Ca2+-activated K+ channels.

The interaction of alpha-human atrial natriuretic peptide (ANP) with salbutamol, sodium nitroprusside and isosorbide dinitrate in human bronchial smooth muscle.

1. Contractions in human bronchial rings evoked by methacholine (10(-6) M) were reversed by single contractions of alpha-human atrial natriuretic peptide (10(-6) M), salbutamol (10(-6) M), sodium nitroprusside (10(-6) M) or isosorbide dinitrate (4.2 x 10(-5) M) and the extent of the relaxations compared. The activity of combinations of ANP with salbutamol, sodium nitroprusside and isosorbide dinitrate were compared with those for each agonist alone. 2. ANP and salbutamol were equipotent in reversing methacholine-evoked contraction and, in combination these agonists evoked an additive response. ANP and sodium nitroprusside also evoked similar degrees of relaxation and were additive, as were ANP and isosorbide dinitrate; however, with isosorbide dinitrate a higher concentration was required to evoke the same degree of relaxation as ANP, sodium nitroprusside or salbutamol. 3. Cumulative concentration-response curves to methacholine (10(-9)-3 x 10(-4) M) were examined in the presence and absence of the above bronchodilator substances, alone and in combination allowing their abilities to protect against contraction to be compared. ANP (10(-6) M) and salbutamol (10(-6) M) each attenuated subsequent contractions evoked by methacholine, an ability not shared with sodium nitroprusside (10(-6) M) or isosorbide dinitrate (4.2 x 10(-5) M). Indeed at lower concentrations of methacholine (< 3 x 10(-7) M), sodium nitroprusside evoked a paradoxical enhancement of methacholine-evoked contractions. 4. In combination, ANP and salbutamol attenuated contractions evoked by methacholine to a significantly greater degree than that seen with either agonist alone, whilst a combination of ANP and sodium nitroprusside evoked no greater effect than that seen with ANP alone. By contrast, isosorbide dinitrate and ANP together evoked a greater inhibition than ANP alone.5 These results suggest that a combination of agents such as ANP and salbutamol evokes a greater effect than either alone, both in reversing and protecting against methacholine-evoked contractions.Such combinations may be of benefit in the treatment of patients, allowing lower doses of drug to be used. Combinations of ANP and isosorbide dinitrate may likewise be of interest; however, the mechanism underlying the enhancement of ANP responses by isosorbide dinitrate requires further study.

Chronic exposure to hypoxia attenuates contractile responses in rat airways in vitro: a possible role for nitric oxide.

We investigated the effect of chronic hypoxia (10% O(2) for 14 days) on airway responsiveness in rats. Chronic hypoxia significantly (P<0. 05, P<0.01, P<0.01, respectively) attenuated contractions evoked by methacholine (10(-9)-3x10(-4) M), endothelin-1 (10(-10)-3x10(-7) M) and potassium chloride (10(-3)-7x10(-2) M) in rat isolated trachea. To investigate this attenuation, we studied the effect of epithelial removal, indomethacin (3x10(-6) M), and L-nitro arginine methyl ester (L-NAME, 10(-4) M), on contractile responses in control and chronically hypoxic rat trachea. Indomethacin did not alter contractions evoked by methacholine or endothelin-1 in control or hypoxic rats. In contrast, epithelial removal and L-NAME both significantly potentiated responses to methacholine and endothelin-1 in trachea from control and chronically hypoxic rats. In separate experiments, tracheal rings were first contracted with methacholine (10(-6) M) and then relaxed, either by the nitric oxide donor sodium nitroprusside or by the beta(2)-adrenoceptor agonist, salbutamol. Sodium nitroprusside was significantly (P<0.001) more effective at reversing induced tone in tracheal rings from chronically hypoxic than control rats. Salbutamol, however, was equally effective in chronically hypoxic and control rats. These results suggest that, in trachea from both control and chronically hypoxic rats, contractile responses to methacholine and endothelin-1 are inhibited by nitric oxide, probably released from the epithelium. The attenuation of contractile responses in airways from chronically hypoxic rats may be due to an enhanced guanylyl cyclase activity and hence, an increased response to nitric oxide.

Effect of angiotensin II on histamine-induced bronchoconstriction in the human airway both in vitro and in vivo.

The renin-angiotensin system is activated in acute severe asthma. Angiotensin II causes bronchoconstriction in mild asthmatics and potentiates methacholine-evoked bronchoconstriction both in vitro and in vivo. To evaluate the effect of angiotensin II on histamine-induced bronchoconstriction, human bronchial rings (n = 6) were obtained from lung tissue at thoracotomy and were prepared in organ baths. Contractions were measured isometrically and cumulative concentration-response curves obtained to angiotensin II alone and to histamine in the presence and absence of threshold concentrations of angiotensin II. Eight asthmatic patients with bronchial hyper-reactivity to histamine were challenged with histamine during intravenous infusion of placebo, angiotensin II 1 ng kg-1 min-1 and angiotensin 2 ng kg-1 min-1 administered in a randomized, double-blind fashion, FEV1 was measured prior to, during the infusion and during the histamine challenge. Angiotensin II (3 x 10(-7)M and 10(-6)M) alone evoked small contractions (< 0.25 g) of human bronchi in vitro, but pre-incubation with threshold concentrations of angiotensin II (10(-7)M, 3 x 10(-7)M and 10(-6)M) had no effect on histamine-evoked contractions. In asthmatic patients, angiotensin II alone had no effect on baseline FEV1 at the low levels infused and did not affect the response to nebulized histamine as measured by the PC20 histamine: Geometric mean (range) PC20 histamine (mg ml-1) screening day 3.58 (1.26-7.75), placebo infusion 2.67 (0.89-9.57), angiotensin II 1 ng kg-1 min-1 2.45 (0.42-6.97) and angiotensin II 2 ng kg-1 3.09 (0.8-10.78). It is concluded that, in contrast to its potentiating effect on methacholine-induced bronchoconstriction, angiotensin II has no effect on histamine-evoked bronchoconstriction in human bronchi in vitro or in vivo.

Microbial sterility testing of oil-formulated bovine somatotropin using Tween 80 dispersion.

The Food and Drug Administration requires that sterile pharmaceutical products be free of viable microorganisms. Sterility testing of pharmaceutical products provides added assurance that the product is sterile. Sterility testing is typically done by inoculating the drug product into microbial growth media followed by visual inspection for growth during incubation for a specified time period. A lack of visual growth indicates that the drug product samples tested were sterile. Formulated Posilac bovine somatotropin consists of protein particles suspended in an oil-based excipient. The product formulation is immiscible in aqueous media due to the excipient's water insolubility and the insolubility of the protein particles at near neutral pH values. Because the formulation is packaged and sold as a sterile product, it is critical that a sensitive microbial sterility test method be used for this key quality test. A sterility test method was developed for Posilac that utilized Tween 80 (i.e. polysorbate 80) as a dispersant. Dispersion of the product using Tween 80 produced a homogeneous suspension of bovine somatotropin particles and oil droplets in the micron size range. The suspension did not appreciably settle out with time, attesting to the homogeneous nature of the mixture. This method was found to be compatible with survival, recovery, and growth from low numbers of the test organisms required by the U.S. Pharmacopeia XXIII as well as from two additional test cultures.

Incidence of benign pathology in patients undergoing hepatic resection for suspected malignancy.

BACKGROUND: Confirming the presence of hepatic or proximal bile duct malignancy pre-operatively remains difficult and some patients may undergo surgical resection for suspected malignant lesions which subsequently turn out to be benign. The aim of this study was to establish whether improvements in pre-operative staging might better identify this patient population. METHODS: Analysis of a prospectively collected database, which has been maintained in our unit since 1988. RESULTS: Of 250 consecutive patients undergoing hepatic resection for presumed malignancy, 18 (7.2%) were shown to have benign pathology. These "false positive" rates were 4 out of 160 (2.5%) resections for colorectal metastases, 4 out of 49 (8.2%) resections for other solid hepatobiliary tumours and 10 out of 41 (24.4%) resections for hilar cholangiocarcinoma. Four of the 18 patients (22%) developed post-operative complications but there was no postoperative mortality. CONCLUSION: Although hepatic resection remains a potentially curative procedure for patients with tumours involving the liver parenchyma or proximal bile ducts, pre-operative confirmation of malignancy remains difficult. Despite appropriate investigation a subset of patients with benign disease will still be subjected to major hepatic resection which should be undertaken in a specialist unit.

Ten-year results of the Press Fit Condylar Sigma total knee replacement.

We report ten-year clinical and radiological follow-up data for the Sigma Press Fit Condylar total knee replacement system (Sigma PFC TKR). Between October 1998 and October 1999 a total of 235 consecutive PFC Sigma TKRs were carried out in 203 patients. Patients were seen at a specialist nurse-led clinic seven to ten days before admission and at six and 18 months, three, five and eight to ten years after surgery. Data were recorded prospectively at each clinic visit. Radiographs were obtained at the five- and eight- to ten-year follow-up appointments. Of the 203 patients, 147 (171 knees) were alive at ten years and 12 were lost to follow-up. A total of eight knees (3.4%) were revised, five for infection and three to change the polyethylene insert. The survival at ten years with an endpoint of revision for any reason was 95.9%, and with an endpoint of revision for aseptic failure was 98.7%. The mean American Knee Society Score (AKSS) was 79 (10 to 99) at eight to ten years, compared with 31 (2 to 62) pre-operatively. Of 109 knee with radiographs reviewed, 47 knees had radiolucent lines but none showed evidence of loosening.

Does obesity influence clinical outcome at nine years following total knee replacement?

A total of 445 consecutive primary total knee replacements (TKRs) were followed up prospectively at six and 18 months and three, six and nine years. Patients were divided into two groups: non-obese (body mass index (BMI) < 30 kg/m(2)) and obese (BMI ≥ 30 kg/m(2)). The obese group was subdivided into mildly obese (BMI 30 to 35 kg/m(2)) and highly obese (BMI ≥ 35 kg/m(2)) in order to determine the effects of increasing obesity on outcome. The clinical data analysed included the Knee Society score, peri-operative complications and implant survival. There was no difference in the overall complication rates or implant survival between the two groups. Obesity appears to have a small but significant adverse effect on clinical outcome, with highly obese patients showing lower function scores than non-obese patients. However, significant improvements in outcome are sustained in all groups nine years after TKR. Given the substantial, sustainable relief of symptoms after TKR and the low peri-operative complication and revision rates in these two groups, we have found no reason to limit access to TKR in obese patients.

Acute Q-fever and history taking--a lesson learned.

A 50-year-old patient underwent a routine primary total hip replacement. Soon after surgery, he developed acute respiratory failure from post-operative sepsis. His condition deteriorated rapidly despite supportive management and he required admission into intensive care unit for assisted ventilation. It took almost one week before the underlying cause of the deterioration was determined to be unrelated to complications of surgery. A diagnosis of Q-fever was made following detailed attention to the clinical history. Appropriate treatment was started and the patient made a full recovery. The diagnosis was confirmed later following discharge from hospital.