Note: Time-gated 3D single quantum dot tracking with simultaneous spinning disk imaging.

We describe recent upgrades to a 3D tracking microscope to include simultaneous Nipkow spinning disk imaging and time-gated single-particle tracking (SPT). Simultaneous 3D molecular tracking and spinning disk imaging enable the visualization of cellular structures and proteins around a given fluorescently labeled target molecule. The addition of photon time-gating to the SPT hardware improves signal to noise by discriminating against Raman scattering and short-lived fluorescence. In contrast to camera-based SPT, single-photon arrival times are recorded, enabling time-resolved spectroscopy (e.g., measurement of fluorescence lifetimes and photon correlations) to be performed during single molecule/particle tracking experiments.

[Surgical treatment of coxofemoral luxation in two cattle]. / Operative Behandlung einer Luxatio ossis femoris bei zwei Rindern.

A three-week-old Holstein Friesian calf and a 20-month-old Jersey heifer were referred to the Department of Farm Animals, University of Zurich, because of coxofemoral luxation. Both animals were moderately lame on the affected hind limb, which was swollen in the hip region and appeared to be adducted and shorter than the contralateral normal hind limb. Radiographs of the affected hips confirmed craniodorsal displacement of the femur. In both animals, traction and open reduction was carried out under general anaesthesia. The joint capsule, which was severely torn, was repaired using suture material or a non absorbable mesh. In the calf, two 4.5-mm screws and washers were placed in the dorsal rim of the acetabulum. Strong non-absorbable suture material of USP 6 in size was placed around each screw and through a pre-drilled hole in the femoral neck and back to the screw in a figure-8 pattern. The sutures were tied and the screws tightened. In the heifer, a non-absorbable mesh was attached to the dorsal acetabular rim using three 4.5-mm cortical screws. The mesh was sutured to the joint capsule at the femoral neck using strong non-absorbable suture material in a simple continuous pattern. Complications were not encountered during the postoperative period. Six months after discharge, both animals were in good general health, although the heifer had mild lameness and muscle atrophy in the operated limb.

The zygomatic bone as a potential donor site for alveolar reconstruction--a quantitative anatomic cadaver study.

The aim of this cadaver study was to evaluate the possibility of using the zygomatic bone as an intraoral bone harvesting donor site and to determine the safety of this harvesting procedure. In addition, the volume of bone material harvested from the zygomatic bone was measured. Twenty fixed adult cadavers were used to yield a total of 40 zygomatic bone harvest sites, from which bone was collected. The volume of bone obtained from the zygomatic harvests was measured with a water displacement method and by compressing the graft into a syringe. The safety of the technique was evaluated by assessing possible encroachment upon the neighbouring structures. After bone harvesting, the zygomatic sites were exposed and evaluated for visible perforations or fractures. Possible damage to the neighbouring tissues was also examined with computed tomography scans at 18 sites in nine cadavers. The average bone graft volume obtained from the zygomatic bone was measured to be 0.53 ml (SD 0.25) with water displacement and 0.59 ml (SD 0.26) with the syringe. The complications in the zygoma included 15 small perforations into the maxillary sinus and 7 perforations into the infratemporal fossa. CT scans showed that bone could be harvested safely without encroaching upon the orbital floor or the surrounding nerves and vessels in the zygoma. The zygomatic bone is a safe intraoral donor site for the reconstruction of small- to medium-sized alveolar defects.

KIN241: a gene involved in cell morphogenesis in Paramecium tetraurelia reveals a novel protein family of cyclophilin-RNA interacting proteins (CRIPs) conserved from fission yeast to man.

In this study, we report cloning, by functional complementation of the KIN241 gene involved in Paramecium cell morphogenesis, cortical organization and nuclear reorganization. This gene is predicted to encode a protein of a novel type, comprising a cyclophilin-type, peptidyl-prolyl isomerase domain, an RNA recognition motif, followed by a region rich in glutamate and lysine (EK domain) and a C-terminal string of serines. As homologues of this protein are present in the genomes of Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana and Homo sapiens, the Kin241p predicted sequence defines a new family of proteins that we propose to call 'CRIP', for cyclophilin-RNA interacting protein. We demonstrate that, in Paramecium, Kin241p is localized in the nucleus and that deletion of some nuclear localization signals (NLSs) decreases transport of the protein into the nucleus. No Kin241-1 protein is present in mutant cells, suggesting that the C-terminal serine-rich region is responsible for protein stability.

Paramecium genome survey: a pilot project.

A consortium of laboratories undertook a pilot sequencing project to gain insight into the genome of Paramecium. Plasmid-end sequencing of DNA fragments from the somatic nucleus together with similarity searches identified 722 potential protein-coding genes. High gene density and uniform small intron size make random sequencing of somatic chromosomes a cost-effective strategy for gene discovery in this organism.

Unusual eustachian tube mass: glomus tympanicum.

SUMMARY: A case of recurrent glomus tympanicum presenting with epistaxis is described. CT and MR imaging revealed a homogeneously enhancing mass extending along the entire course of the eustachian tube, with a portion protruding into the nasopharynx. Glomus tumors tend to spread along the path of least resistance and may extend into the eustachian tube. The unique imaging appearance should place a glomus tumor high on the list of differential diagnoses.

ND9P, a novel protein with armadillo-like repeats involved in exocytosis: physiological studies using allelic mutants in paramecium.

In Paramecium, a number of mutants affected in the exocytotic membrane fusion step of the regulated secretory pathway have been obtained. Here, we report the isolation of one of the corresponding genes, ND9, previously suspected to encode a soluble protein interacting with both plasma and trichocyst membranes. Nd9p is a novel polypeptide that contains C-terminal Armadillo-like repeats. Point mutations were found in the first N-terminal quarter of the molecule and in the last putative Armadillo repeat, respectively, for the two thermosensitive mutants, nd9-1 and nd9-2. The different behaviors of these mutants in recovery experiments upon temperature shifts suggest that the N-terminal domain of the molecule may be involved in membrane binding activity, whereas the C-terminal domain is a candidate for protein-protein interactions. The nonsense nd9-3 mutation that produces a short N-terminal peptide has a dominant negative effect on the nd9-1 allele. We show here that, when overexpressed, the dominant negative effect can be produced even on the wild-type allele, suggesting competition for a common target. We suggest that Nd9p could act, like some SNARE proteins, at the membrane-cytosol interface to promote membrane fusion.

An indexed genomic library for Paramecium complementation cloning.

Recent pioneering work opened the way for cloning genes in Paramecium by functional complementation of mutants. We present here the construction and pilot utilization of a new indexed library of Paramecium macronuclear DNA. The library is made of 61,440 clones containing inserts mostly between 6 and 12 kilobases. It has already allowed the complementation cloning of four new genes, and this library has proven to be very useful for rapid hybridization cloning.

Positional localization: three-dimensional transthoracic echocardiographic techniques for the measurement of cardiac mass, volume, and function.

An accurate and reproducible determination of cardiac volume and mass is important for the selection and timing of therapeutic interventions. Quantitative three-dimensional echocardiography has evolved to provide these measurements with the use of a noninvasive, readily available, and inexpensive technique. We introduce and review the principle of positional localization as well as the clinical application of this technique for the measurement of cardiac volume and mass.

Left and right atrial volume by freehand three-dimensional echocardiography: in vivo validation using magnetic resonance imaging.

INTRODUCTION: Measurement of left and right atrial size is important for the management of arrhythmias, valvular and congenital heart disease. We have demonstrated that freehand three-dimensional (3D) echocardiography is more accurate and reproducible than two-dimensional (2D) echocardiography for measurement of left ventricular mass and volume. However, no prior study has validated the accuracy of freehand 3D for the determination of left or right atrial volume. METHODS: End-systolic (maximum) left and right atrial volumes were determined in 21 volunteer patients and normal subjects by one, two, and freehand 3D transthoracic echocardiography and compared to volumes obtained by gradient recalled magnetic resonance imaging. Three-dimensional echocardiographic determination of atrial volume was obtained using an acoustic spatial locator, a line-of-intersection display, and a surface reconstruction algorithm. Two-dimensional echocardiographic atrial volumes were obtained from apical biplane images of the left atrium and an apical single plane image of the right atrium using a summation of disks method. One-dimensional (ID) estimates of left atrial volume were determined by cubing the M-mode ID antero-posterior dimension obtained on the parasternal long axis view. RESULTS: An excellent correlation was Obtained between freedhand 3D echocardiography and magnetic resonce imaging (MRI) for the left atrium (r = 0.90, SEE=9.6 ml) and for the right atrium (r = 0.91, SEE = 8.8 ml) with a small bias (left atrium 5.25 ml, right atrium 12.06 ml) and narrow limits of agreement (left atrium 22.14 ml, right atrium 25.54 ml). Two-dimensional echocardiography correlated less well (left atrium r = 0.87, SEE = 10.23 ml, right atrium r = 0.79, SEE = 19.74 ml), and had a higher bias (left atrium 14.46 ml, right atrium 8.99 ml) and larger limits of agreement (left atrium 24.37 ml, right atrium 41.16 ml). One-dimensional estimates of left atrial volume correlated poorly with magnetic resonance determined left atrial volume (r = 0.80, SEE = 6.61 ml) and had unacceptably high bias (45.09 ml) and limits of agreement (35.52 ml). Interobserver variability was lowest for 3D echocardiography (left atrium 7.2 ml, 11%, right atrium 8.7 ml, 16%). CONCLUSIONS: Freehand 3D echocardiography using the line of intersection display for guidance of image positioning and a polyhedral surface reconstruction algorithm is a valid, accurate, reproducible method for determining left and right atrial volume in humans that is comparable to magnetic resonance imaging and is superior to current ID and 2D echocardiographic techniques.

Further evaluation of intensified and increased total dose of cyclophosphamide for the treatment of primary breast cancer: findings from National Surgical Adjuvant Breast and Bowel Project B-25.

PURPOSE: In 1989, the National Surgical Adjuvant Breast and Bowel Project initiated the B-22 trial to determine whether intensifying or intensifying and increasing the total dose of cyclophosphamide in a doxorubicin-cyclophosphamide combination would benefit women with primary breast cancer and positive axillary nodes. B-25 was initiated to determine whether further intensifying and increasing the cyclophosphamide dose would yield more favorable results. PATIENTS AND METHODS: Patients (n = 2,548) were randomly assigned to three groups. The dose and intensity of doxorubicin were similar in all groups. Group 1 received four courses, ie, double the dose and intensity of cyclophosphamide given in the B-22 standard therapy group; group 2 received the same dose of cyclophosphamide as in group 1, administered in two courses (intensified); group 3 received double the dose of cyclophosphamide (intensified and increased) given in group 1. All patients received recombinant human granulocyte colony-stimulating factor. Life-table estimates were used to determine disease-free survival (DFS) and overall survival. RESULTS: No significant difference was observed in DFS (P =.20), distant DFS (P =.31), or survival (P =.76) among the three groups. At 5 years, the DFS in groups 1 and 2 (61% v 64%, respectively; P =. 29) was similar to but slightly lower than that in group 3 (61% v 66%, respectively; P = 08). Survival in group 1 was concordant with that in groups 2 (78% v 77%, respectively; P =.71) and 3 (78% v 79%, respectively; P =.86). Grade 4 toxicity was 20%, 34%, and 49% in groups 1, 2, and 3, respectively. Severe infection and septic episodes increased in group 3. The decrease in the amount and intensity of cyclophosphamide and delays in therapy were greatest in courses 3 and 4 in group 3. The incidence of acute myeloid leukemia increased in all groups. CONCLUSION: Because intensifying and increasing cyclophosphamide two or four times that given in standard clinical practice did not substantively improve outcome, such therapy should be reserved for the clinical trial setting.

Evidence for Clostridium perfringens enterotoxin (CPE) inducing a mitogenic and cytokine response in vitro and a cytokine response in vivo.

We investigated some immunogenic properties of Clostridium perfringens enterotoxin (CPE) in vitro using murine J774A macrophages (MPhi) and in vivo using Swiss Webster (SW) mice. CPE was a potent mitogen in vitro, where cell proliferation increased with CPE concentration. CPE was nonmitogenic when MPhi were concurrently incubated with CPE and interferon gamma (IFN-gamma). MPhi incubated in the presence of CPE induced the synthesis of interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha) and interferon-gamma (IFN-gamma), but not interleukin-2 (IL-2). In vivo, CPE induced a pro-inflammatory cytokine response with striking production of IFN-gamma, IL-1, and IL-6. Regardless of route of CPE entry, serum cytokine levels generally peaked within 1 h of administration and were maintained for 4-8 h. Although CPE engenders an intense immune response during toxicosis, the toxin does not appear to be a superantigen. Death from CPE-induced shock appears to result from various interrelating immunological mechanisms.

Cost-effectiveness of assertive community treatment versus standard case management for persons with co-occurring severe mental illness and substance use disorders.

OBJECTIVE: To determine the cost-effectiveness of Assertive Community Treatment (ACT) in comparison to Standard Case Management (SCM) for persons with severe mental illness and substance use disorders. DATA SOURCES AND STUDY SETTING: Original data on the effectiveness and social costs of ACT and SCM that were collected between 1989 and 1995. Seven community mental health centers in New Hampshire provided both types of treatment. STUDY DESIGN: Persons with schizophrenia, schizoaffective disorder, or bipolar disorder and a concurrent substance use disorder were randomly assigned to ACT or SCM and followed for three years. The primary variables assessed were substance use, psychiatric symptoms, functioning, quality of life, and social costs. DATA COLLECTION METHODS: Effectiveness data were obtained from interviews at six-month intervals with persons enrolled in treatment and with their service providers. Social cost and service utilization data came from client reports; interviews with informal caregivers; provider information systems and Medicaid claims; law enforcement agencies; courts; and community service providers. PRINCIPAL FINDINGS: Participants in both groups showed significant reductions in substance use over time. Focusing on quality of life and substance use outcomes, ACT and SCM were not significantly different in cost-effectiveness over the entire three-year study period. Longitudinal analyses showed that SCM tended to be more efficient during the first two years but that ACT was significantly more efficient than SCM during the final year of the study. CONCLUSIONS: In an adequately funded system, ACT is not more cost-effective than SCM. However, ACT efficiency appears to improve over time.

Homology-dependent maternal inhibition of developmental excision of internal eliminated sequences in Paramecium tetraurelia.

Thousands of single-copy internal eliminated sequences (IESs) are excised from the germ line genome of ciliates during development of the polygenomic somatic macronucleus, following sexual events. Paramecium IESs are short, noncoding elements that frequently interrupt coding sequences. No absolutely conserved sequence element, other than flanking 5'-TA-3' direct repeats, has been identified among sequenced IESs; the mechanisms of their specific recognition and precise elimination are unknown. Previous work has revealed the existence of an epigenetic control of excision. It was shown that the presence of one IES in the vegetative macronucleus results in a specific inhibition of the excision of the same element during the development of a new macronucleus, in the following sexual generation. We have assessed the generality and sequence specificity of this transnuclear maternal control by studying the effects of macronuclear transformation with 13 different IESs. We show that at least five of them can be maintained in the new macronuclear genome; sequence specificity is complete both between genes and between different IESs in the same gene. In all cases, the degree of excision inhibition correlates with the copy number of the maternal IES, but each IES shows a characteristic inhibition efficiency. Short internal IES-like segments were found to be excised from two of the IESs when excision between normal boundaries was inhibited. Available data suggest that the sequence specificity of these maternal effects is mediated by pairing interactions between homologous nucleic acids.

Effects of prenatal cocaine exposure on the mesocorticolimbic dopamine system: an in vivo microdialysis study in the rat.

Microdialysis studies were conducted on prenatally saline-treated and prenatally cocaine-treated rats, either as pups (10-30 days old) or young adults (40-190 days old), to study the effects of prenatal cocaine exposure on the mesolimbic dopamine (DA) system. In the n. accumbens of saline-treated rats, basal dialysate concentrations of DA were similar in pups and adults; however, the levels of DA metabolites, DOPAC, HVA, and the serotonin metabolite, 5-HIAA, were markedly lower in pups. In pups, prenatal cocaine exposure led to basal dialysate levels of DA in the n. accumbens that were twice control levels; however, there was no difference in response to a period of intermittent tail pinch or an acute injection of cocaine (20 mg/kg). In the adult, basal levels of DA, DOPAC, HVA and 5-HIAA in n. accumbens did not differ across prenatal treatments. However, in prenatally cocaine-treated adults a cocaine injection led to an enhanced rise in extracellular DA compared to controls. In frontal cortex of adult rats, basal levels of DA, DOPAC and HVA did not differ across prenatal treatments; however, basal levels of 5-HIAA in this region were significantly elevated in prenatal-cocaine rats. No group differences were observed in the frontal cortex in response to either tail pinch or cocaine. Thus prenatal cocaine exposure produces an increase in basal extracellular DA in the n. accumbens of pups which returns to normal with aging. While this initial difference normalizes, prenatal cocaine exposure induces other persistent changes in adulthood.

Identification and treatment of children's mental health problems by primary care providers: a critical review of research.

This article critically reviews research studies on the identification and treatment of children's mental health problems by primary care providers. Using a valid and reliable instrument to guide the review, the investigators examined the methods and findings of all 13 studies published on the topic from 1979 to 1994. Overall, primary care providers appeared to underidentify mental health problems among children and adolescents, especially those whose emotional impairments were not overtly severe. Large proportions of youngsters identified as having mental health problems did not receive needed treatment.

Fadrozole HCL (CGS-16949A) versus megestrol acetate treatment of postmenopausal patients with metastatic breast carcinoma: results of two randomized double blind controlled multiinstitutional trials.

BACKGROUND: Breast cancer patients with prior response to endocrine therapy achieve subsequent benefit from additional endocrine therapies. The efficacy and safety of an aromatase inhibitor, fadrozole HCL, were compared with megestrol acetate in post menopausal patients who had disease progression after receiving antiestrogen therapy either for metastatic disease or as adjuvant therapy. METHODS: In 2 multiinstitutional prospective trials, 683 postmenopausal patients were randomized to receive either fadrozole HCL, 1 mg twice daily, or megestrol acetate, 40 mg 4 times daily, in a double blind fashion after progression on first-line hormonal therapy. Objective response rates, time to progression, survival and safety of the two regimens were compared. RESULTS: Results of intent-to-treat analyses are presented in this study. No significant differences were detected between the two treatment groups in time to progression, objective response rates, duration of response, and survival in either trial. There were no clinically meaningful differences between the treatment groups in the incidence and severity of adverse experiences, except that weight gain, fluid retention, and dyspnea were observed in more patients in the megestrol acetate group compared with those receiving fadrozole HCL, whereas nausea and vomiting were observed in more patients in the fadrozole HCL group compared with those receiving megestrol acetate. CONCLUSIONS: Fadrozole HCL was as efficacious as megestrol acetate in postmenopausal patients with metastatic breast carcinoma after one hormonal therapy. Adverse experiences were mild with both therapies, but megestrol acetate was associated wiht a higher frequency of weight gain, fluid retention and dyspnea, whereas fadrozole HCL was associated with a higher frequency of nausea and vomiting.