A dsbA mutant of Pseudomonas syringae exhibits reduced virulence and partial impairment of type III secretion.

Abstract To identify virulence genes of P. syringae pv. tomato strain DC3000 we screened for mutants with reduced virulence on its plant hosts, Arabidopsis thaliana and tomato. We isolated a Tn5-insertion mutant that exhibited reduced virulence on both hosts. Further characterization showed that this mutant carried a single Tn5 insertion in the dsbA gene, which encodes a periplasmic disulphide bond-forming protein. In addition to reduced virulence, the dsbA mutant exhibits mucoid colony morphology, loss of fluorescence, decreased motility, and a reduced growth rate in culture. The dsbA mutant is able to multiply in A. thaliana and tomato plants, trigger the hypersensitive response on tobacco and elicit Pto-mediated resistance in tomato, indicating that type III secretion occurs in this background. However, type III secretion appears to function with reduced efficiency in the dsbA mutant, as type III-dependent secretion of HrpZ and AvrRpt2 is impaired. These findings indicate that while the dsbA gene is required for multiple cellular functions in P. syringae, type III secretion in P. syringae is only partially dependent on dsbA.

Analysis of adhesion molecules in myocardial biopsies of cardiac allografts and coronary artery disease with CABG.

Interactions of leukocytes with vascular endothelium are important components of inflammation tissue reactions and have been implicated in cardiac transplant rejection and demonstrated to be mediated by cell adhesion molecules (CAM's). The expression of ICAM-1, VCAM-1 and E-selectin in human myocardium is variable and little is known about the expression of LFA-1 and Mac-1 during allograft rejection. This study investigated these CAM's in myocardial biopsies of transplanted hearts (HTX) and of coronary artery disease eligible for coronary artery bypass grafting (CABG) as non-inflammatory controls and explicitly examines vascular endothelium, cardiomyocytes and infiltrating cells. Immunohistochemistry was performed using the APAAP-method and directing specific mouse anti-human monoclonal antibodies against ICAM-1 (CD54), VCAM-1 (CD106), E-selectin (CD62E), alpha-LFA-1 (CD11a), alpha-Mac-1 (CD11b), alpha-p150/95 (CD11c) and the beta2-integrin chain (CD18). CD18, LFA-1 (CD11a), Mac-1 (CD11b) and p150/95 (CD11c) were markedly expressed on infiltrating immunocytes in HTX compared to CABG where no expression of beta2-integrins was observed. Cardiac allografts demonstrated a strong expression of ICAM-1 on vascular endothelium and on infiltrating cells. ICAM-1 was not detected on cardiomyocytes. In CABG a weak expression of ICAM-1 was observed on endothelial cells but not on myocytes. VCAM-1 was expressed on vascular endothelium and perivascular infiltrating cells in HTX but not in CABG. VCAM-1 was not found to be expressed on myocytes. There was no evidence for the presence of E-selectin in any of our biopsy specimens. Our study shows that the study of cell adhesion molecules adds to the pathophysiological understanding of inflammation after transplantation in cardiac disease. This offers a potential for the development of diagnostic tools and new therapeutic strategies.

[The late cardiac sequelae after mantle-field irradiation. The results in Erlangen's patient caseload]. / Kardiale Spätfolgen nach Mantelfeldbestrahlung. Ergebnisse im Erlanger Patientenkollektiv.

PURPOSE: We have retrospectively evaluated the cardiac function in patients after mantle-irradiation for Hodgkin's disease. PATIENTS AND METHODS: Forty-three patients, mean age 39.8 +/- 13 years, who had been treated with mantle-irradiation from 1979 through 1984 at the University of Erlangen-Nürnberg, were examined. All of them were in first remission at 5 to 11 years (mean 8.1 years) after radiotherapy (n = 24) or combined modality treatment (n = 19). Mantle-irradiation had been administered through equally weighted anterioposterior-posterioanterior portals with 5 fractions of 2 Gy per week up to a total mediastinal dose of 41.8 +/- 7 Gy (including boost). The examination program included anamnesis, physical examination, Doppler echocardiography and ergometry. Hundred and twenty-two non-irradiated volunteers and cardiological patients were used as control group. RESULTS: None of the patients suffered from clinical symptoms. Pericardial thickening was present in 26%. The left ventricular end-systolic diameter (31.3 +/- 5.5 mm, normal value 26 to 42 mm) and the thickness of the left ventricular posterior wall (8.1 +/- 1.8 mm, normal value 6 to 11 mm) were within the normal range. The left ventricular ejection fraction was also normal (67.1 +/- 8% in patients versus 67.0 +/- 8% in control subjects). Evaluation of diastolic parameters, however, revealed significant changes. The isovolumic relaxation time was significantly decreased as compared to the control group (38.8 +/- 17 ms versus 50.8 +/- 21 ms, p < 0.05). The same we found for the shortening fraction (33.6 +/- 6% versus 38.1 +/- 6%, p < 0.05). The most significant changes were found after combined modality therapy, especially after radiotherapy and anthracycline-based chemotherapy. 30% of the patients were tachycardic (pulse > 100/min) at rest. At ergometry, the frequency of signs of ischemia (5%), conduction disturbances (2%), and rhythm disturbances (7%) was not elevated. CONCLUSIONS: In this retrospective investigation, patients after mantle irradiation with modern techniques showed only minimal cardiologic abnormalities within the first 10 years after treatment. The most sensitive parameters were the isovolumic relaxation times. The clinical relevance of such findings remains to be defined. Pathophysiologically, our findings (decreased relaxation time plus tachycardia) support the theory that myocardial damage after radiotherapy may result in subsequent increased beta-receptor density as proposed by Schultz-Hector et al. [21] on the basis of experimental data.

[Extended reversible global left ventricular contraction dysfunction with symptomless coronary system as effect of cytostatic therapy with 5-fluorouracil]. / Ausgedehnte reversible globale linksventrikuläre Kontraktionsstörung als Folge einer zytostatischen Therapie mit 5-Fluorouracil bei unauffälligem Koronarsystem.

CASE REPORT: We report on a young female patient suffering from anal-cancer who received simultaneous radiochemotherapy and developed a "hibernating myocardium" during continuous infusion of 5-FU. CONCLUSION: In literature the incidence of cardiac complications caused by 5-FU-therapy is found to be between 1% and 10%. Patients with coronary heart disease have a four-fold higher risk. Possible pathogenic mechanisms are spasm of coronary artery, direct cardiotoxicity, immunological reactions and disturbance of the coagulation system.

[Angiography follow-up after transluminal angioplasty of coronary bypass grafts]. / Angiographische Verlaufskontrollen nach transluminaler Angioplastie von koronaren Bypass-Grafts.

Between January 1979 and October 1991, percutaneous transluminal angioplasty of stenosed or occluded coronary bypass grafts was attempted 180 times in 146 patients (180 lesions in 157 bypass grafts); 6/157 grafts were internal mammary grafts. The procedure was successful in 129/157 grafts (82%) and in 151/180 lesions (84%). Failures occurred almost exclusively in recanalization attempts. Cardiac complications occurred in 4/146 patients (2.7%). Three patients developed an acute myocardial infarction, another patient died after acute occlusion of a native vessel dilated during the same procedure. In successful attempts the severity of stenosis was reduced from 87 +/- 10% to 33 +/- 15%. 113/129 successfully dilated grafts had at least one (mean 2.7) control angiogram. 54/113 (48%) showed recurrence after a mean follow up of 6 months. An additional 15 grafts showed late restenosis in a second control angiogram (mean follow-up 23 months). The total restenosis rate was 61%. Restenoses were dilated again one to six times (mean 1.9) with comparable success and recurrence rate. Two patients died during the sixth angioplasty. Finally, 32/129 (25%) grafts were occluded or presumably occluded, and 97/129 (75%) were angiographically confirmed open without restenosis. Thus, angioplasty of bypass grafts is an alternative to a repeat revascularization surgery. The acute results are comparable to the results of angioplasty in native coronary arteries. The restenosis rate is high. One has to be aware of late restenosis. Restenosis can be dilated repeatedly with a comparable success rate and with no significant increase in restenosis rate.

Nested PCR for specific detection and rapid identification of human picornaviruses.

A nested PCR for the detection and rapid identification of human picornaviruses is described. Enteroviruses and rhinoviruses were amplified with the same set of four primers from the 5'-noncoding region. The nested primers allowed the detection of far less than 1 PFU in diluted virus stocks without Southern blot hybridization. In patients with neurological disorders (mainly aseptic meningitis), 43% of 37 specimens (11 of 21 cerebrospinal fluid specimens, 2 of 10 serum specimens, and 3 of 6 stool specimens) were positive by PCR. A total of 21% (10 of 47 specimens) of heart biopsy specimens from patients with dilative cardiomyopathy were PCR positive, whereas 3% (2 of 70 specimens) of control biopsy specimens from patients with coronary artery disease were PCR positive. PCR-amplified fragments from 27 of 29 clinical isolates and 14 of 28 patient samples were successfully serotyped by restriction enzyme digestion. Two specimens were further investigated by direct sequencing of PCR products, leading to the identification of a poliovirus type 3 isolate with a sequence that was highly divergent from previously published sequences.

PTCA in elderly patients: acute results and long-term follow-up.

Acute results and long-term follow-up of percutaneous transluminal coronary angioplasty (PTCA) in 125 patients aged 75 to 90 years (79 men; 46 female; mean age 78 +/- 3 years), with mainly unstable angina (102 patients) are reported. Successful PTCA was achieved in 96 out of 108 patients (89%); occlusions could be reopened in eight out of 17 patients (47%). Thirty-eight procedures were multiple vessel or multiple lesion PTCA, so that the lesion-related success rate of PTCA was higher (91%). Major complications occurred in seven patients (5.6%) including one procedure-related death. In-hospital mortality was 3% and concerned exclusively patients with unstable angina and unsuccessful procedure. At a mean follow-up of 27 +/- 16 months (2 to 82 months) 13 additional patients had died: 10 from cardiovascular causes, three from cancer. In the meantime, three patients underwent non-fatal myocardial infarction and three elective bypass surgery; 26 patients had repeat PTCA. Eighty-seven of the 93 surviving patients with successful primary procedure and reintervention if necessary, were in anginal class I or II; only six were in class III. We conclude that the success rate of PTCA in elderly patients is comparable to that in younger patients, and that a successful procedure leads to continued relief of symptoms.

[Recurrent myxoma]. / Rezidivmyxom.

In a now 53-year-old woman, who suffered from recurrent syncope and dizziness, a biatrial myxoma originating from the interatrial septum was resected nearly 4 years ago, and the resulting septal defect was closed by a Dacron patch. In the symptomless patient the recurrent myxoma was revealed by echocardiography. The left atrial myxoma was surgically removed without repeat heart catheterization. This case underlines the importance of regular, echocardiographic follow-up examinations in patients in whom a myxoma has been excised. In case of suspicious findings, an additional transesophageal examination or nuclear magnetic resonance imaging should be performed.

[Percutaneous transluminal coronary angioplasty in patients with clearly restricted left ventricular pump function]. / Perkutane transluminale Koronarangioplastie bei Patienten mit deutlich eingeschränkter linksventrikulärer Pumpfunktion.

Among nearly 2000 consecutive PTCA-patients 42 (36 male, six female; mean age 60 +/- 11 years) had ejection fractions < or = 35% (mean 30 +/- 5%). 34 patients suffered from unstable and eight from stable angina. All had previous myocardial infarction and of these 23 had recent myocardial infarction. Four patients had prior coronary bypass surgery and one had undergone aneurysmectomy. 31 of 42 (= 74%) patients had multiple vessel disease (stenoses > 50%). Successful procedure was achieved in 35 of 42 patients (= 83%). The lesion-related success rate of PTCA was 89%, of recanalization 60%. Six procedures were multiple vessel PT-CA. Major complications occurred in two of 42 patients (one myocardial infarction, one emergency bypass operation). The in-hospital mortality was 2.4%. Follow-up angiography was performed in 22 patients and showed restenoses in nine cases. All patients underwent repeat coronary angioplasty, of these one patient had postprocedural myocardial infarction. The ejection fraction had meanwhile significantly improved from 29 +/- 5% to 36 +/- 7%. Clinical follow-up after 18 +/- 14 months (two to 53 months) was available in 39 of 40 patients, who were discharged from hospital without major complications. 29 patients were free of angina or anginal class II, one had a late cardiac transplant. Five patients had died of cardiac and one of noncardiac causes. Thus, PTCA in patients with severely depressed ventricular function shows acceptable acute results, which are comparable to those of routine angioplasty, continued symptomatic improvement can be achieved. However, the late outcome is significantly worse than in patients with normal left ventricular function.

Temporal and spatial control of the mother-cell regulatory gene spoIIID of Bacillus subtilis.

Gene expression during endospore formation in Bacillus subtilis is compartmentalized between the mother-cell and forespore chambers of the sporangium, which follow separate pathways of cellular differentiation. The earliest acting regulatory gene so far identified in the mother-cell line of gene expression is spoIIID, whose product is required for the transcription of the composite gene (sigK) encoding the mother-cell RNA polymerase sigma-factor sigma K and for the chromosomal rearrangement that gives rise to the composite gene. Here we report the nucleotide sequence of spoIIID and studies on the temporal, spatial, and genetic control of its expression during sporulation. We show that the deduced spoIIID gene product, a 93-residue-long polypeptide, is a previously identified transcription factor that is known to activate the promoter for the sigK gene in vitro. Expression of spoIIID is largely confined to the mother-cell chamber of the sporangium and is turned on at, or shortly before, the time (hour 3 of sporulation) that the mother-cell chromosome is rearranged and transcription of the sigK gene commences. This gene expression depends strongly on the sporulation sigma-factor sigma E and partially on the spoIIID gene product, itself. We conclude that the timing and compartmentalization of the rearrangement and transcription of the sigK gene and, hence, of subsequent gene activation in the mother cell, are, in part, direct consequences of the temporal and spatial control of spoIIID gene expression.

[Hemodynamic study of the development of tolerance in intravenous nitrate therapy]. / Hämodynamische Untersuchung zur Toleranzentwicklung unter intravenöser Nitrattherapie.

UNLABELLED: To investigate possible tolerance development under an intravenous treatment with nitrates, 22 patients with coronary artery disease were randomly assigned to receive either 4 mg/h of ISDN (n = 12) or placebo (n = 10) and were given additional 10 mg of ISDN or placebo after 23 h. Pulmonary artery pressures (PAP), cardiac output, and heart rate were registered before, and 4 h, 22.5 h, and 24 h after the beginning of treatment. At baseline both groups were similar with regard to pulmonary artery diastolic pressure (PADP) at rest and at comparable work load. Each patient of the placebo group showed identical PAPs at work. ISDN led to a 42-59% decrease of PADP at rest and 29-37% decrease at comparable work load. After 22.5 h a statistically insignificant reduction of ISDN effect was observed which could be reversed by additional ISDN p.o. A detailed analysis of the ISDN group showed seven patients with a persistently lowered PADP, (table; see text) whereas five patients demonstrated a partial diminuation of the ISDN effect. No patient showed a complete tolerance. In the placebo group blood pressure did not show any change during the treatment period (143 +/- 21/84 +/- 10 mmHg vs 137 +/- 17/79 +/- 10 mmHg), whereas ISDN infusion resulted in a continuously lowered blood pressure (150 +/- 22/83 +/- 11 mmHg vs 125 +/- 15/72 +/- 9 mmHg (p less than 0.001). Cardiac output and heart rate were similar under ISDN and placebo treatment. CONCLUSION: Continuous infusion of 4 mg of ISDN/h over 24 h and an additional dose of 10 mg of ISDN after 23 h provided a significant reduction in PADP, blood pressure and rate pressure product over 24 h at rest and during exercise.

[Imaging of aortocoronary bypasses with intravenous digital subtraction angiography]. / Darstellung aortokoronarer Bypasses mit der intravenösen digitalen Subtraktionsangiographie.

We examined 24 patients with 52 coronary bypass grafts, an average of 18 months after their respective operations. During the course of 1 week, a coronary angiography and a digital subtraction angiography (DSA) incorporating an intravenous injection of contrast medium were performed. Conventional coronary angiograms showed 40 bypasses as being open, ten as being occluded, and two could not be displayed at all. With the aid of digital angiography, 50 out of 52 bypasses could be classified as either open or occluded. In 44 out of 52 bypasses, DSA and coronary angiogram results were identical. Using DSA, three out of ten angiographically occluded bypasses were falsely diagnosed as being open and three out of 40 open bypasses as occluded. Two bypasses could not be interpreted due to poor picture quality. In the diagnosis "open bypass" the degree of both sensitivity and specificity subsequently amounted to 92.5%, and 70% in the diagnosis "occluded bypass". The distal part of the bypasses, as well as the proximal and distal part of the anastomoses, could not be evaluated for the most part. Furthermore, on account of the comparatively inferior quality of the pictures, detection of bypass stenosis is not reliable using digital subtraction angiography. Intravenous digital subtraction angiography may therefore serve as a screening method in the evaluation of coronary bypass grafts.

[Magnetic resonance tomography of the postoperative lumbar spine. A comparison of the MR versus CT images in recurrent intervertebral disk prolapse]. / Kernspintomographie der operierten Lendenwirbelsäule. Bildvergleich MR versus CT bei Bandscheibenrezidiven.

In the present study, 40 patients who had undergone disc surgery were examined by high resolution CT and MR for possible recurrence of the disc prolapse and the results are compared. It appears that high resolution spinal MR, using its various tissue parameters provides no new insights into possible recurrence of a disc prolapse. As is the case with CT, the investigator must also evaluate other morphological and clinical factors. In spite of this, MR can be useful in the investigation of abnormal spines.

Development and regression of right heart ventricular hypertrophy: biochemical and morphological aspects.

Male Wistar rats were exposed, in a hypobaric chamber, to a simulated altitude of 6000 m for up to four weeks. The animals quickly developed pulmonary hypertension with an important media hypertrophy of the pulmonary arteries, followed by severe right heart hypertrophy (cor pulmonale). Right heart hypertrophy is evident in three morphologically and biochemically definable stages. In stage 1 (1st-2nd week) a manifest thickening of heart muscle cells develops due to increased protein synthesis. In stage 2 (2nd-3rd week) one can find, in the regular biochemical composition of heart muscle, an activation of mitochondrial ATPase, a multiplication of mitochondria, a proliferation of interstitial cells and an increase in interstitial volume. In stage 3 (3rd-4th week) the hypertrophied myocardium exhibits signs of biochemical and morphological decompensation. Besides a loss of myofibrils and a reduction in mitochondrial ATPase, DNA and protein concentrations sink to subnormal values. Only myocardium from stage 1 of hypertrophy shows complete reversibility after cessation of hypobaric conditions, but not so in stage 3. Parallel with the developing cor pulmonale, the animals also react with a small hypertrophy of the left heart ventricle. This concomitant growth persists under normobaric conditions, too. These investigations document that growth of myocardium under extreme conditions shows a phasic development. Severe forms of myocardial hypertrophy do not always seem to be reversible.

Myocardial biopsy in patients with hypertrophic cardiomyopathy: correlations between morphologic and clinical parameters and development of myocardial hypertrophy under medical therapy.

Left ventricular biopsies from 38 patients with hypertrophic cardiomyopathy (HOCM 28, HNCM 10) were investigated to evaluate possible correlations between morphological and clinical parameters. No correlation was found between the degree of myocardial hypertrophy (muscle cell diameter), nuclear size of the myocytes, fibrous tissue content and various clinical data such as pressure gradient, left ventricular end-diastolic pressure, Sokolow index and heart volume. In 11 patients with HOCM, a second biopsy was performed after medical therapy (verapamil, n = 9; propranolol, n = 2) over 33 +/- 12 months. Increasing myocardial hypertrophy (cell diameter 16.2 +/- 4.4 mu vs. 20.3 +/- 4.2 mu) was observed in all 11 patients. The interstitial fibrous tissue content increased from 5.7 +/- 6.3 to 12.7 +/- 6.8%. The volume fraction of myofibrils decreased (48.8 +/- 2.7 vs. 43.6 +/- 5.3%). The morphological changes were observed regardless of the clinical outcome which was improved in four, unchanged in five and worsened in two cases. The underlying hypertrophic process in HCM seems to be slowly progressive in most patients and cannot be influenced by medical treatment.

[Coronary perfusion in acute vascular occlusion within the scope of transluminal coronary angioplasty]. / Koronarperfusion bei akutem Gefässverschluss im Rahmen der transluminalen Koronarangioplastik (TCA).

The procedure of coronary perfusion during transluminal coronary angioplasty (TCA) is described. By using guide wires of a length of 300 cm it is possible to exchange a balloon catheter for a perfusion catheter in acute vascular occlusion. Arterial blood is withdrawn and correspondingly supplied distal to the occlusion to meet the myocardial need for sufficiently oxygenated blood. Ischemia resolves, resulting in the normalization of left ventricular function and resolution of subjective complaints. This procedure can maintain myocardial oxygen supply until emergency coronary bypass graft surgery or repeat angioplasty. It might help to reduce mortality secondary to transluminal coronary angioplasty.

[Biopsy and autopsy frequency of myocarditis]. / Bioptische und autoptische Häufigkeit der Myokarditis.

Mononuclear infiltrations indicative for myocarditis were found in 3 out of 171 left ventricular biopsies from patients with cardiomyopathies (1.2%). One patient had a normal EF, one had a minimally and one a markedly reduced ventricular function. Among 5,804 consecutive necropsies myocarditis was diagnosed in 308 cases (5.3%). 1.3% were septic in origin while mononuclear infiltrations were found in 3.7% and special morphology in 0.3%. Combined detailed analysis both of clinical and autopsy data could be performed in 84 patients who had died in our own hospital. 82 patients had severe additional diseases determining clinical symptoms and course. Myocarditis without other underlying diseases was found in 2 patients only. Infiltrations were widespread and diffuse in both ventricles and atria in these cases. Myocardial inflammation could not be diagnosed from clinical parameters (X-ray, symptoms, ECG) in any case. Our bioptical data demonstrate that myocarditis does not play a major role in the pathogenesis of dilated cardiomyopathies. Diffuse myocarditis of clinical relevance rarely occurs in adults while focal myocardial infiltrations can be found in a majority of diseases. Reliable clinical symptoms indicative for inflammatory myocardial disease could be found neither in cases with concomitant nor in patients with diffuse myocarditis.

[2 families with dilatative cardiomyopathy]. / Zwei Familien mit dilatativer Kardiomyopathie.

In two brothers each of two families congestive cardiomyopathy was diagnosed. Left ventricular biopsy showed individually differing degrees of hypertrophy of the myocardial cells together with endocardial fibrosis and circumscribed interstitial fibrosis. In family I clinical symptoms appeared at the beginning of the 4th decade, conduction disturbances being predominant. One brother died after rapid progression of the disease within one year. The other has been treated as an outpatient for 9 years; he received a pacemaker implant half a year ago. In family II initial symptoms appeared in the middle of the 6th decade, progressive heart dilation and insufficiency being the predominant characteristics. In the course of 7 years one brother has developed cardiac decompensation whereas the other brother's condition has remained nearly unchanged for 4 years. The degree of myocardial changes in left ventricular biopsies correlated with the clinical course.

[Reversibility of akinetic segments in coronary heart disease]. / Untersuchungen zur Reversibilität akinetischer Segmente bei der koronaren Herzkrankheit.

Akinetic wall segments not exhibiting contractions following nitroglycerin administration or in a post-extrasystolic beat are usually considered to consist of scar tissue; i.e. even by re-established or improved blood supply following aorto-coronary bypass surgery no functional improvement is expected. In the present study, the pre- and postoperative ventriculograms (RAO projection) of 24 patients undergoing bypass surgery were analyzed. Ventriculography was routinely performed following sublingual nitroglycerin and a post-extrasystolic contraction. In each patient the akinetic segment had received a bypass graft which was found to be patent on reangiography. In 7 of 24 patients (29%) the formerly akinetic segment exhibited improved contraction postoperatively; in 17 patients the segment remained akinetic. Global ejection fraction rose in the group of patients with improved akinesia from 47 +/- 10 to 65 +/- 10% (p less than 0.05). In the patients with unchanged contraction pattern, ejection fraction was found to be 56 +/- 12% prior to surgery and 54 +/- 16% after surgery (n.s.). The increase in ejection fraction was more pronounced in those patients showing improvement of anterior wall akinesia (from 39 to 72%) than it was in patients exhibiting improved inferior wall akinesis (from 54 to 59%). According to these findings, the regional ejection fraction was found to be higher postoperatively in patients with former anterior wall akinesis (78%) than in those showing inferior wall contraction abnormalities (49%). End-diastolic and end-systolic left ventricular volume changes postoperatively did not reach statistical significance, although end-systolic volume showed a clear trend to decrease (preoperative: 114 +/- 54 ml/1.73 m2; postoperative: 79 +/- 29 ml/1.73 m2; n.s.).(ABSTRACT TRUNCATED AT 250 WORDS)