A methodology for markerless genetic modifications in Azotobacter vinelandii.

AIMS: Efficient manipulation of multiple regions within a genome can be improved by counter-selection approaches. In this work, we sought to develop a method to manipulate Azotobacter vinelandii using a counter-selection approach based on the presence of the pyrF gene. METHODS AND RESULTS: A background uracil auxotroph of A. vinelandii was first constructed by deleting the pyrF gene coding orotidine-5'-phosphate decarboxylase. The pyrF gene and promoter were also incorporated together with an antibiotic marker to create a selection and counter-selection cassette to shuttle into various plasmids. The constructed cassette could then be removed using a plasmid lacking the pyrF gene via counter-selection resulting from the production of 5-fluorouracil. The process could be repeated multiple times using the same procedure for selection and counter-selection. Following completion, the pyrF gene may be reintroduced to the genome in its original location, leaving a completed strain devoid of any antibiotic markers. CONCLUSIONS: Utilization of the pyrF gene for counter-selection is a powerful tool that can be used effectively to make multiple gene deletions in A. vinelandii. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the successful application of a counter-selection approach to yield markerless genetic modifications to A. vinelandii, which should be of interest for a range of applications in this important model bacterium.

Nonlinear domain-wall velocity enhancement by spin-polarized electric current.

The interaction between a dc spin-polarized electric current and a magnetic domain wall in a Permalloy nanowire was studied by high-bandwidth scanning Kerr polarimetry. The full functional dependence of wall velocity on electric current and magnetic field is presented. With the pinning potential nulled by a field, current-induced velocity enhancements exceeded 35 m/s at a current density of approximately 6 x 10(11) A/m(2). This large enhancement, more than 10 times that found in pinning-dominated experiments, results in part from an interaction that is nonlinear in current and independent of current direction.

Condensed-phase effects on the structural properties of C6H5CN-BF3 and (CH3)3CCN-BF3: IR spectra, crystallography, and computations.

Condensed-phase effects on the structure and bonding of C(6)H(5)CN-BF(3) and (CH(3))(3)CCN-BF(3) are illustrated by a variety of results, and these are compared to analogous data for the closely related complex CH(3)CN-BF(3). For the most part, the structural properties of C(6)H(5)CN-BF(3) and (CH(3))(3)CCN-BF(3) are quite similar, not only in the gas phase but also in the solid state and in argon matrices. However, the structures do change significantly from medium to medium, and these changes are reflected in the data presented below. Specifically, the measured crystallographic structure of C(6)H(5)CN-BF(3) (s) has a B-N distance that is 0.17 A shorter than that in the equilibrium gas-phase structure obtained via B3LYP calculations. Notable differences between calculated gas-phase frequencies and measured solid-state frequencies for both C(6)H(5)CN-BF(3) and (CH(3))(3)CCN-BF(3) were also observed, and in the case of (CH(3))(3)CCN-BF(3), these data implicate a comparable difference between solid-state and gas-phase structure, even in the absence of crystallographic results. Frequencies measured in argon matrices were found to be quite similar for both complexes and also very near those measured previously for CH(3)CN-BF(3), suggesting that all three complexes adopt similar structures in solid argon. For C(6)H(5)CN-BF(3) and (CH(3))(3)CCN-BF(3), matrix IR frequencies differ only slightly from the computed gas-phase values, but do suggest a slight compression of the B-N bond. Ultimately, it appears that the varying degree to which these systems respond to condensed phases stems from subtle differences in the gas-phase species, which are highlighted through an examination of B-N distance potentials from B3LYP calculations. The larger organic substituents appear to stabilize the potential near 1.8 A, so that the structures are more localized in that region prior to any condensed-phase interactions. As a result, the condensed-phase effects on the structural properties of C(6)H(5)CN-BF(3) and (CH(3))(3)CCN-BF(3) are much less pronounced than those for CH(3)CN-BF(3).

Total synthesis of the immunosupressant (-)-pironetin (PA48153C).

[reaction: see text]. Total synthesis of the immunosuppresant pironetin has been achieved by a synthetic route in which the connections between starting materials and the desired structure are readily discerned. Key steps include a diastereoselective Lewis acid mediated crotylstannane aldehyde addition, a highly selective Lewis acid promoted Mukaiyama aldol reaction, an anti-selective SmI2 reduction of a beta-hydroxyketone, and finally a lactone annulation reaction.

Evaluating the effects of functional communication training in the presence and absence of establishing operations.

We conducted functional analyses of aberrant behavior with 4 children with developmental disabilities. We then implemented functional communication training (FCT) by using different mands across two contexts, one in which the establishing operation (EO) that was relevant to the function of aberrant behavior was present and one in which the EO that was relevant to the function of aberrant behavior was absent. The mand used in the EO-present context served the same function as aberrant behavior, and the mand used in the EO-absent context served a different function than the one identified via the functional analysis. In addition, a free-play (control) condition was conducted for all children. Increases in relevant manding were observed in the EO-present context for 3 of the 4 participants. Decreases in aberrant behavior were achieved by the end of the treatment analysis for all 4 participants. Irrelevant mands were rarely observed in the EO-absent context for 3 of the 4 participants. Evaluating the effectiveness of FCT across different contexts allowed a further analysis of manding when the establishing operations were present or absent. The contributions of this study to the understanding of functional equivalence are also discussed.

A versatile preparation of alpha,beta-unsaturated lactones from homoallylic alcohols.

[formula: see text] A new method for the synthesis of alpha,beta-unsaturated lactones from beta-acetoxy aldehydes by reaction with the lithium enolate of methyl acetate was developed. The reaction is relatively insensitive to structural changes in the aldehyde substrates. The process was extended to the synthesis of five-ring lactones from alpha-acetoxy aldehydes. Experimental evidence regarding the mechanism of this one-pot transformation was obtained. The observations are consistent with a pathway involving an initial aldol condensation with subsequent acyl migration, lactonization, and beta-elimination and not an enolate equilibration-aldol mechanism.

Conversion of cyclodextrin into high-amylose starch of low molecular mass by means of cyclodextrin glucanotransferase.

alpha-Cyclodextrin (CD) was converted by Bacillus macerans cyclodextrin glucanotransferase into highly insoluble high-amylose starch with a low average degree of polymerization (56-73), in yields as high as 78% over a wide range of temperatures (25-70 degreesC). Ability to undergo this conversion was highly concentration-dependent. gamma-CD was also convertible in good yield; however, beta-CD was relatively resistant to conversion. Degrees of polymerization and the percentages of amylose in the high-amylose products were estimated from spectrophotometric measurements on their iodine complexes. A plausible mechanism for conversion of CD into starch is proposed.

Child and adolescent traumatic brain injury: correlates of disruptive behaviour disorders.

A record review focused on children and adolescents, with a history of traumatic brain injury (TBI), who were consecutively admitted to a brain injury clinic in which all new patients are psychiatrically evaluated. Correlates of post-injury oppositional defiant disorder/conduct disorder (ODD/CD) and post-injury attention deficit hyperactivity disorder (AHD) were investigated. Subjects who developed ODD/CD following TBI, when compared to subjects without a lifetime history of the disorder, had significantly more impaired family functioning, showed a trend toward a greater family history of alcohol dependence/abuse and suffered a milder TBI. In contrast, there were no variables which discriminated between subjects who developed ADHD following injury and those with no lifetime history of ADHD. It is difficult to determine whether ODD, CD and ADHD occurring after TBI in the patient is related to the TBI, directly or indirectly. Appropriate clinical assessment requires consideration of the important mediating role of family functioning, severity of injury and family psychiatric history.

Child and adolescent traumatic brain injury: correlates of injury severity.

A record review focused on children and adolescents, with a history of traumatic brain injury, who were consecutively admitted to a brain injury clinic in which all new patients are psychiatrically evaluated. Significant correlates of severity of injury in the cognitive, education and communication domains of functioning included Performance IQ but not Verbal IQ nor standardized ratings of language or learning disability. Current organic personality syndrome (OPS) but not attention deficit hyperactivity disorder or oppositional defiant disorder/conduct disorder diagnostic status was significantly related to severity. In conclusion, the findings in this referred sample are similar to prospective studies indicating that Performance IQ appears sensitive in reflecting brain damage. The finding linking OPS to severity of injury is not surprising. This is because OPS is a diagnosis which is dependent on the clinician's judgment of the likelihood that the organic factor is etiologically related to a defined behavioural syndrome. The diagnosis therefore requires a clinical judgment that the threshold of severity of a presumed organic etiological factor has been reached.

Expanding the provider pool to increase access to services.

PIP: To meet the health care needs of low-income, uninsured, and underserved people in the US, mid-level health care personnel such as nurse practitioners (NPs) and physician assistants (PAs) are providing a range of services which used to be provided only by physicians. Greater access to services, high-quality provider-client interaction, and an emphasis upon health education and prevention have been cited by participants in many studies as reasons why clients are so satisfied with NP-provided services and why widespread support exists for using them to perform many types of procedures. One example of the use of mid-level health care personnel is a project launched in November 1996 in Alaska, Idaho, Oregon, and Washington which trains NPs and PAs how to perform no-scalpel vasectomies (NSVs). This project, a collaborative effort of AVSC, the US Public Health Services, and the National Association of Nurse Practitioners in Reproductive Health, was launched to improve access to vasectomy services among low-income and underserved men. The Center for Health Training in Seattle, WA, coordinated the training. 12 clinicians were trained to perform NSVs in 1997, and trainees' skills in performing NSVs will be evaluated during the 8- to 12-month period following training. Reactions to the project have thus far been very positive.^ieng

Child and adolescent traumatic brain injury: psychiatric findings from a paediatric outpatient specialty clinic.

A record review focused on children and adolescents, with a history of traumatic brain injury, who were consecutively admitted to a brain injury clinic in which all new patients are psychiatrically evaluated. The development of a 'novel' psychiatric disorder (not present before injury) occurred in 76% (38/50) of the cohort and was correlated significantly with family psychiatric history and family function, but not with severity of injury, preinjury psychiatric status, intellectual/educational functioning, or socioeconomic status. Psychiatric consultation is often necessary in this paediatric population even though much of the psychopathology, particularly following mild injury, may not be directly related to brain trauma.

Colonoscopic evaluation of rectal bleeding in young adults.

The role of colonoscopy in the evaluation of blood loss per rectum has been studied extensively in older patients but not in the younger adult population. The objective of this study was to review the diagnostic yield of colonoscopy in patients 40 years of age or younger with passage of minimal amounts of bright red blood per rectum or occult blood positive stools. Retrospectively, 5924 colonoscopic examinations were reviewed. All patients, 40 years of age or younger, with rectal bleeding as the only indication for colonoscopy were selected. A total of 280 patients met these criteria and 59 (21%) had significant findings. Polyps were present in 25 (8.9%) of patients. Other findings included colitis, diverticular disease, and angiodysplastic lesions present in 8.6 per cent, 2.1 per cent, and 1.1 per cent, respectively. Adenocarcinoma was present in one patient (0.03%). These results suggest that colonoscopy in patients 40 years of age or younger with rectal bleeding may be justified because significant findings may be present in up to 21 per cent of patients.

Quality assurance in the clinical genetics setting-Report of a workshop.

Participants in a panel discussion sponsored by thead hoc Quality Assurance Committee of the National Society of Genetic Counselors discuss early efforts to develop and implement quality assurance instruments at institutional, state, and regional levels. Uniform guidelines and self-assessment tools can help genetic counselors and clinical geneticists to provide the best possible care to individuals affected by genetic diseases. Further work will be needed to address aspects not covered here and to assure that guidelines allow for creative variation among professionals and centers. An interdisciplinary approach to such standard-setting is recommended.

Tetrasomy 9p: tissue-limited idic(9p) in a child with mild manifestations and a normal CVS result. Report and review.

Supernumerary isochromosomes resulting in autosomal tetrasomy are rare and have been described only for 12p, 18p, and 9p. Nineteen previous cases of tetrasomy 9p have been reported, and in 6 cases, tissue-specific mosaicism was implied with the i(9p) cell line present exclusively or predominantly in blood. We report on an infant who had apparently normal chromosomes (46,XY) on CVS. He was referred for genetic evaluation because of mild developmental delay and minor anomalies. In 75% of blood cells he had an extra isodicentric 9p chromosome (pter-->q12-->pter). The interpretation of tetrasomy 9p was confirmed by elevated GALT activity. No tetrasomy 9p cells were seen in 100 skin fibroblasts. This case demonstrates the tissue specific mosaicism in tetrasomy 9p which rendered the anomaly undetectable by CVS. It also demonstrates the mild end of the clinical spectrum associated with tetrasomy 9p.

Utilization of amniocentesis and chorionic villus sampling by South Carolina women 35 years of age and older.

An increase in utilization of prenatal diagnosis was observed from 1985 to 1986 in South Carolina. The overall rate of 39.9% for 1986 is comparable with other areas of the U.S. Utilization was correlated with geographic residence, race, and referral source. While there was considerable variation in prenatal diagnostic test utilization between counties in South Carolina, overall utilization rates were reasonably high and continued to increase from 1985 to 1986. It will be interesting to see what effect CVS has on overall utilization rates as this new procedure becomes more established throughout the state.

Immunological aspects of type 1 and 2 diabetes mellitus.

IDDM occurs predominantly among individuals being class II antigen HLA-DR 3 and/or 4 positive, while NIDDM is not associated with HLA-D. Although the HLA-DR 3 or 4 specificities are prerequisites for IDDM to develop, their high frequencies (about 60%) in the background population preclude tissue typing as a predictive test, underlined by the observation that less than 50% of monozygotic twins are concordant for IDDM. The presence of a number of immune abnormalities argues that the causes of IDDM may be sought in an altered immune reaction against antigens present in the pancreatic B cells and/or in the environment. The majority of IDDM patients of short duration show both cellular and humoral autoimmunity against the pancreatic B cells. Similar phenomena may be observed in patients initially diagnosed as NIDDM and treated with oral hypoglycemic agents. It has been speculated that these patients have a retarded form of IDDM. It is possible that the combination of specific Class II antigen molecule(s) and an invading antigen (virus, bacterium, chemical etc.) presented to the immune system triggers the formation of effector cells such as B lymphocytes and cytotoxic T lymphocytes which also cross-react with the pancreatic B cells. Multiple exposures to this or related antigens throughout several years may eventually lead a sufficient loss of pancreatic B cells to cause insulin dependence.

Isolated regional lymph node dissection: morbidity, mortality and economic considerations.

Between 1971-1979, 330 consecutive isolated regional lymph node dissections (RLNDs) were performed as therapeutic procedures for metastatic disease, at the University of Louisville Affiliated Hospitals and the Ellis-Fischel State Cancer Hospital in Columbia, Missouri. This retrospective analysis includes 133 radical neck RLNDs, 87 axillary RLNDs, and 110 superficial groin RLNDs. All accessioned cases were elective and were performed as isolated procedures, discrete from resection of contiguous or remote organs. No patients received preoperative irradiation, chemotherapy or immunotherapy. Forty-eight per cent of the 330 RLND procedures resulted in some form of postoperative complication. However, 91% of the incurred morbidity was localized to the operative site and was related to serum collection and/or flap necrosis. The occurrence of postoperative complications for each RLND site resulted in a prolongation of the patients' hospital stays by a mean of 9 days, and was most extended for the superficial groin RLND by a mean of 11 days. Nine patients (3%) died. These data for morbidity and mortality rates, as well as the implicit economic impact, represent substantial factors in the utilization of elective RLND.

Colonoscopic diagnosis of angiodysplasias of the gastrointestinal tract.

We have reviewed our experience with colonoscopy in the diagnosis of angiodysplastic lesions of the colon. In 14 of the patients in our series, the lesions in the colon were diagnosed colonoscopically, confirming arteriographic findings in many but being the only diagnostic modality facilitating the diagnosis in three of the patients. Results of our experience indicate that colonoscopy is an effective means of diagnosing or confirming the diagnosis of angiodysplastic lesions of the right colon. It should be part of the evaluation along with superior and inferior mesenteric angiography of every patient with occult gastrointestinal tract bleeding or iron deficiency anemia, or both, for whom no diagnosis can be made with standard barium contrast techniques.