Genomic Analysis of a Pathogenicity Island in Uropathogenic Escherichia coli CFT073: Distribution of Homologous Sequences among Isolates from Patients with Pyelonephritis, Cystitis, and CatheterAssociated Bacteriuria and from Fecal Samples

Urinary tract infection is the most frequently diagnosed kidney and urologic disease and Escherichia coli is by far the most common etiologic agent. Uropathogenic strains have been shown to contain blocks of DNA termed pathogenicity islands (PAIs) which contribute to their virulence. We have defined one of these regions of DNA within the chromosome of a highly virulent E. coli strain, CFT073, isolated from the blood and urine of a woman with acute pyelonephritis. The 57,988-bp stretch of DNA has characteristics which define PAIs, including a size greater than 30 kb, the presence of insertion sequences, distinct segmentation of K-12 and J96 origin, GC content (42.9%) different from that of total genomic DNA (50.8%), and the presence of virulence genes (hly and pap). Within this region, we have identified 44 open reading frames; of these 44, 10 are homologous to entries in the complete K-12 genome sequence, 4 are nearly identical to the sequences of E. coli J96 encoding the HlyA hemolysin, 11 encode P fimbriae, and 19 show no homology to J96 or K-12 entries. To determine whether sequences found within the junctions of the PAI of CFT073 were common to other uropathogenic strains of E. coli, 11 probes were isolated along the length of the PAI and were hybridized to dot blots of genomic DNA isolated from clinical isolates (67 from patients with acute pyelonephritis, 38 from patients with cystitis, 49 from patients with catheter-associated bacteriuria, and 27 from fecal samples). These sequences were found significantly more often in strains associated with the clinical syndromes of acute pyelonephritis (79%) and cystitis (82%) than in those associated with catheter-associated bacteriuria (58%) and in fecal strains (22%) (P < 0.001). From these regions, we have identified a putative iron transport system and genes other than hly and pap that may contribute to the virulent phenotype of uropathogenic E. coli strains.     

Control of established experimental allergic encephalomyelitis by inhibition of tumor necrosis factor (TNF) activity within the central nervous system using monoclonal antibodies and TNF receptor-immunoglobulin fusion proteins

Tumor necrosis factor (TNF) activity was inhibited during the development of actively-induced, chronic relapsing experimental allergic encephalomyelitis (CREAE) in Biozzi AB/H mice, using a mouse TNF-specific (TN3.19.12) antibody and bivalent human p55 and p75 TNF receptor-immunoglobulin (TNFR-Ig) fusion proteins. The development of disease could be inhibited when repeated doses of antibody were administered prior to the anticipated onset. It has now also been shown that a therapeutic effect is evident even when antibody is administered after the onset of clinical signs, further indicating an important role for TNF in pathogenic effector mechanisms in CREAE. Although biologically-active TNF was not detected in the circulation, TNF-α was detected in lesions within the central nervous system (CNS). This suggested that the CNS may be the main site for TNF-specific immunomodulation and was supported by the observation that intracranial injection was significantly more potent than that administered systemically, for both antibody and TNFR-Ig fusion proteins. The fusion proteins were as effective as antibody at doses 10—100-fold lower than that used for antibody, reflecting their higher neutralizing capacity in vitro. Although treatment was not curative and relapse inevitably occurred in this model if treatment was not sustained, the data indicate that anti-TNF immunotherapy, especially within the CNS, can inhibit CREAE and may, therefore, be useful in the control of human neuroimmunological diseases.     

Molecular mechanism of kNBC1–carbonic anhydrase II interaction in proximal tubule cells

We have recently shown that carbonic anhydrase II (CAII) binds in vitro to the C-terminus of the electrogenic sodium bicarbonate cotransporter kNBC1 (kNBC1-ct). In the present study we determined the molecular mechanisms for the interaction between the two proteins and whether kNBC1 and CAII form a transport metabolon in vivo wherein bicarbonate is transferred from CAII directly to the cotransporter. Various residues in the C-terminus of kNBC1 were mutated and the effect of these mutations on both the magnitude of CAII binding and the function of kNBC1 expressed in mPCT cells was determined. Two clusters of acidic amino acids, L⁹⁵⁸DDV and D⁹⁸⁶NDD in the wild-type kNBC1-ct involved in CAII binding were identified. In both acidic clusters, the first aspartate residue played a more important role in CAII binding than others. A significant correlation between the magnitude of CAII binding and kNBC1-mediated flux was shown. The results indicated that CAII activity enhances flux through the cotransporter when the enzyme is bound to kNBC1. These data are the first direct evidence that a complex of an electrogenic sodium bicarbonate cotransporter with CAII functions as a transport metabolon.     

Infection of immunodeficient horses with Sarcocystis neurona does not result in neurologic disease

Equine protozoal myeloencephalitis is a progressive neurologic disease of horses most commonly caused by infection with the apicomplexan parasite Sarcocystis neurona. Factors affecting neuroinvasion and neurovirulence have not been determined. We investigated the pathogenesis of infection with S. neurona in horses with severe combined immune deficiency (SCID). Two immunocompetent (IC) Arabian horses and two Arabian horses with SCID were infected orally with 5 x 10(5) sporocysts of S. neurona. Four IC horses and one SCID horse were infected intravenously (i.v.) with 5 x 10(8) merozoites of the WSU-1 isolate of S. neurona. Despite prolonged parasitemia and persistent infection of visceral tissues (skeletal muscle, cardiac muscle, lung, liver, and spleen) as demonstrated by PCR and culture, SCID horses did not develop neurologic signs after oral or i.v. infection. S. neurona was undetectable in the neuronal tissues of SCID horses by either PCR, immunohistochemistry, or culture. In contrast, although parasitemia was undetectable in orally infected IC horses and of only short duration in i.v. infected IC horses, four of six IC horses developed neurologic signs. S. neurona was detectable by PCR and/or culture of neural tissue but not visceral tissue of IC horses with neurologic disease. Infected SCID horses are unable to clear S. neurona from visceral tissues, but the infection does not result in neurologic signs; in contrast, IC horses rapidly control parasitemia and infection of visceral tissues but frequently experience neuroinvasion and exhibit clinical signs of neurologic disease.     

The educational impact of ACGME limits on resident and fellow duty hours: a pre-post survey study.

PURPOSE: To assess the educational impact of Accreditation Council for Graduate Medical Education resident work-hour limits implemented in July 2003. METHOD: All trainees in all 76 accredited programs at two large teaching hospitals were surveyed between May and June 2003 (before work-hour reductions) and then between May and June 2004 (after work-hour reductions) about hours, education, and fatigue. Based on changes in weekly duty hours, 13 programs experiencing substantial reduction in hours were classified into a reduced-hours group. Differences in assessments of educational endpoints before and after policy implementation by trainees in the reduced-hours group were compared with those in other programs to control for potential temporal trends, using two-way ANOVA with interaction. RESULTS: The number of respondents was 1,770 (60% response rate). The reduced-hours group reported a significant decrease in time spent directly caring for patients (from 48.5 to 42.3 mean h/wk, P = 0.03), but the volume of important clinical experiences, including procedures, was preserved, as was the sense of clinical preparedness. On 22 questions related to educational quality and adequacy, only three differences in differences were significant, with the reduced-hours group reporting a relative increase in opportunities for research, decrease in quality of faculty teaching, and decrease in educational satisfaction. The percentage of trainees reporting frequent negative effects of fatigue dropped more in the reduced-hours programs than in the other programs (P < 0.05). CONCLUSION: This study shows that it may be possible to reduce residents' hours--and the perceived adverse impact of fatigue--while generally preserving the self-assessed quality, quantity, and outcomes of graduate medical education.     

Idiopathic congenital central hypoventilation syndrome: the next generation

In the present study, we sought to identify genetic variation in the metabotropic glutamate receptor 3 (GRM3) gene, which has been mapped to chromosome 7q21.1-q21.2 [Scherer et al., 1996] and might contribute to genetic predisposition to schizophrenia and/or bipolar affective disorder. Using single-strand conformation analysis (SSCA), we screened the complete coding sequence as well as adjacent splice sites of the GRM3 gene in a sample of 46 bipolar affective and 46 schizophrenic patients. We detected three sequence variants: a rare C/T substitution at nucleotide position +885 (T209T), a C/T substitution at nucleotide position +2130 (Y624Y), and a more common C/T substitution at nucleotide position +1131 (A291A). The occurrence of the +1131C/T variant was investigated in a sample of bipolar affective patients (n=283), schizophrenic patients (n=265), and ethnically matched controls (n=227). We observed a significant overrepresentation of the +1131T allele in schizophrenic patients when compared to controls (P=0.0022). This finding was followed up in an independent sample of schizophrenic patients (n=288) and controls (n=162) and 128 schizophrenic trios but could not be confirmed. It is therefore unlikely that this variant plays a major role in predisposing to schizophrenia and/or bipolar affective disorder at least in the German population.     

Internal medicine and general surgery residents' attitudes about the ACGME duty hours regulations: a multicenter study.

PURPOSE: To assess internal medicine and general surgery residents' attitudes about the effects of the Accreditation Council for Graduate Medical Education duty hours regulations on medical errors, quality of patient care, and residency experiences. METHOD: In 2005, the authors surveyed 200 residents who trained both before and after duty hours reform at six residency programs (three internal medicine, three general surgery) at five academic medical centers in the United States. Residents' attitudes about the effects of the duty hours regulations on the quality of patient care, residency education, and quality of life were measured using a survey instrument containing 19 Likert scale questions on a scale of 1 to 5. Survey responses were compared using the Student's t-test. RESULTS: The response rate was 80% (159 residents). Residents reported that whereas fatigue-related errors decreased slightly, errors related to reduced continuity of care significantly increased. Additionally, duty hours regulations somewhat decreased opportunities for formal education, bedside learning, and procedures, but there was no consensus that graduates would be less well trained after duty hours reform. Residents, particularly surgical trainees, reported improvements in quality of life and reduced burnout. CONCLUSIONS: Residents in medicine and surgery had similar opinions about the effects of duty hours reform, including improved quality of life. However, resident opinions suggest that reduced fatigue-related errors have been offset by errors related to decreased continuity of care and that the quality of the educational experience may have declined. Quantifying the degree to which regulating duty hours affected errors related to discontinuity of care should be a focus of future research.     

Integrated sibling-parent group intervention to improve sibling knowledge and adjustment to chronic illness and disability

OBJECTIVE: To evaluate an integrated group intervention for siblings and parents designed to increase sibling understanding of and adjustment to chronic illness and developmental disability (CI/DD). METHODS: Fifty-four well siblings (ages 8-13 years) and their parents were recruited through hospital-based and community agencies serving children with CI/DD. Measures of sibling knowledge, sibling adjustment to the disorder, sibling connectedness, and sibling global behavioral functioning were collected before and after the intervention. A subsample of 20 families completed a 3-month follow-up to assess maintenance of results. RESULTS: Sibling knowledge of the child's disorder and sibling connectedness increased, while sibling reports of negative adjustment to the disorder and parent reports of sibling global behavioral functioning decreased significantly from pre- to posttreatment for both boys and girls, regardless of the type of diagnostic condition. Improvements in sibling knowledge, connectedness, and behavioral problems maintained at 3-month follow-up. Parent satisfaction with the program was high. CONCLUSIONS: Results support the future conduct of more controlled evaluation of the integrated sibling and parent group intervention model to improve sibling knowledge of and adjustment to CI/DD.     

Healing of osteochondral osteotomies after fixation with a hydroxyapatite-buffered polylactide. A histomorphometric and radiographic study in rabbits

The tissue response of subchondral bone to a biodegradable fixation device manufactured in the shape of a screw and made of polylactide with a hydroxyapatite buffer were implanted through the articular surface of the intercondylar portion of the distal rabbit femur. One screw was implanted per animal. The screws had a core diameter of 3.2 mm and an outer diameter of 4.5 mm. At insertion, the implants were cut flush with the articular surface. After follow-up times of 8 and 16 weeks, the specimens were examined radiographically and histomorphometrically. The intact contralateral femur served as a control for comparison. Only minimal signs of degradation of the polymer could be seen in the histologic specimens. These implant degradation sites were commonly areas of new bone formation adjacent to the screw implant. A brim of repair tissue was formed at the entrance and exit of the implant channel. The width of the repair tissue from the tissue-implant boundary towards the center of the entrance hole varied greatly between the specimens, from 80 to 750 microm. In most specimens this bridging tissue consisted of newly formed bone and undifferentiated mesenchymal tissue. Degenerative chondrocyte clustering occurred in the pre-existing cartilage within a 400 microm wide zone from the tissue-implant interface into the recipient tissues. Some new-bone formation was seen to envelop the implant in all specimens, but the fractional osteoid formation surface of the trabeculae was only significantly higher in the screw-implanted 16-week specimens, when compared to the non-operated contralateral controls. Although the bony osteotomy was invariably healed in all specimens with good implant integration, the quality and quantity of the reparative tissue of the articular cartilage near the screw hole was variable. This study showed that large polylactide implants, which are buffered with hydroxyapatite show benign tissue responses and good implant osteointegration when implanted in bone. They may be suitable for fixation of small bone fractures. However, insertion through intra-articular surfaces may require further improvement of the implant material to avoid the degenerative repair processes seen in this study.     

Geneticists' views on science policy formation and public outreach

Though much research about the public's views of scientists, genetic research and its moral, ethical, and social implications exists, little has been done to investigate how scientists view their own role(s) in public discussions and policy formation related to genetic research and technologies. We interviewed 20 academic geneticists in the United States about their perceptions of the roles they and others (e.g., professional societies, the public, ethicists, and elected officials) do and should play in the formation of science policy, the communication of science to the public, and the public discussions of moral and ethical issues raised by scientific advances. The participants in our study thought that scientists should be more actively involved in public outreach and science policy formation, but frequently they felt ill-equipped and unsupported by their peers and institutions to pursue these activities. Furthermore, many were skeptical of or did not trust elected officials--who they consider uninformed about the issues and too driven by political agendas--to formulate sound science policy. They do, however, have faith in the ability of scientific societies to influence policy effectively, and some thought that societies should play a larger role, both in science policy and as a liaison between scientists and the public. Finally, participants offered suggestions for increasing the involvement and influence of scientists in science-policy formation and public discourse.